Transcription Initiation in the Histidine Operon of Salmonella typhimurium * Abstract .
Evidence is presented for two natural transcription initiators positioned internally in the histidine operon of Salmonella typhimurium .
They were detected using highly polar S. typhimurium his mutants as recipients for F ' Escherichia coli his episomes .
These intergeneric complementation tests provide a sensitive method for the detection of other initiators and of terminators .
That at least part of the operator for the his operon lies distal to the trans-cription initiator is suggested by transduction analysis of a new highly polar mutant described in this paper .
The his operon of S. typhimurium consists of an operator region followed by nine genes encoding the enzymes for histidine biosynthesis .1 Generally , deprivation of histidine from growing cultures results in a sequential expression of the genes which proceeds from the operator end and which yields a coordinate elevation of the his enzymes .1 Repression of such derepressed cells also proceeds sequentially2 and is coordinate .1 An alteration to the pattern of derepression yielding simultaneous but still apparently coordinate effects may be obtained by increasing the pool of one-carbon fragments in these cells .
' These data , the direction of polarity effects among his mutants ,1 4 and studies of the size of his mRNA , 1 5 suggest this operon is transcribed into a single species of polycistronic mRNA .
A single promoter at the operator end is presumed to serve as transcription initiator .
Several deletion mutants have been reported which lack both hisO and one or more of the proximal structural genes .1 Some of these are highly polar and have been interpreted as lacking the his promoter .
Our isolation of a new polar mutant , his-3601 , led us to re-examine the properties of such mutants .
We report evidence for the existence of two transcription initiators positioned internally in the his operon .
These initiators are normally present , but may have insignificant expression in the normal system .
In addition to the natural transcription initiators , mutationally derived initiators whose level of function are not determined have been obtained in the first structural gene .
Detection of the initiators makes use of complementation with appropriate E. coli F ' his mutants .
This intergeneric complementation occurs in the virtual absence of recombination , and is suggested as a general method for the detection of initiators and terminators .
Mapping of his-3601 indicates that the his operon promoter , P1 , lies proximal to at least part of the his operator .
With the exceptions given below , media and bacteriological methods were essentially as described previously .6 9 Transductions for phagesensitive recombinants were done using the integration deficient variant of P22 , L4 .10 His-8601 is a spontaneously arising mutant isolated after mutant enrichment by recycling in penicillin .
It was selected as a histidinol nonutilizing mutant in a strain containing a constitutive mutation , his01242 , and amber mutation , hisG2187 .
The resultant strain is termed SC209 .
A varient of SC209 containing an amber suppressor for mutation hisG2187 was constructed by first screening for pro- .
This was then mated as recipient for F'pro + lac - ( amber ) for growth in the absence of proline .
Phage L4 was propagated on a strain containing an amber suppressor for hisG2187 ; the episome-containing strain was then transduced for utilization of lactose and finally was cured of its episome .
Designation and properties of these strains appear in Table 1 .
Other Salmonella strains are described elsewhere : his mutants ; ; 6 11 , 12 F'lac - strains .
'' ' F ' T80 his + gnd + ( gnd indicates the gene for expression of 6-phosphogluconate dehydrogenase ) originally indicated to be an episome bearing Salmonella genes , was subsequently recognized as containing E. coli genetic information .
' 4 Episomal mutants other than E. coli hisF-and hisC-were obtained through the courtesy of J. Roth and colleagues .
Additional auxotrophic markers where required were obtained after penicillin screening following treatment with either N-methyl-N ' - nitro-N-nitrosoguanidine or diethylsulfate .
With two exceptions , episomes were established in donor strains containing chromosomal deletions of the relevant his genes .
This avoided the possibility of growth in subsequent tests as a result of mobilization of his + DNA from the chromosome of cells donating the F ' factor .
F'hisA - was used as obtained in a background of trp - , pur - , his-612 .
F'hisDa was maintained by its gnd + property in a background of his-660 which had been mutated to lack gluco-nate utilization .
F'hisDb and F'hisDab were used as obtained in a background of trp - , pur - , his-612 .
All other episomes were established in strains bearing his-644 , trpABE13O .
For all episomal tests apart from F'hisD - , episomes were established in the recipients by growth upon plates containing histidinol as a histidine source ; purified clones were then streaked for isolated colonies on minimal media .
The same results were obtained by plating donor strains directly onto lawns of the recipients using minimal plates .
In tests using F'hisD - mutants the donor strains were plated directly on lawns of the recipients .
Enzyme assays of histidinol phosphate phosphatase were performed as described by Ames , Garry , and Herzenberg '' in the presence of 10-2 Ml Mg + + following a suggestion of B. N. Ames .
The enzyme `` isomerase '' was assayed as described by Margo-lies and Goldberger .
`` 6 Protein determinations were by the Lowry method as described in reference 15 .
Intergeneric complementation : In a number of systems , episomal tests of intraspecies complementation yield positive results within 24 hours ; low-level complementation may be obscured by the appearance of recombinant clones within two days , e.g. , reference 17 .
Available data indicate the his operons in S. typhimurium and E. coli may have the same gene sequence ,12 but recombination in intergeneric crosses is rare , ' 8 including crosses in the his region .
' 4 ' 19 This is presumably a consequence of an underlying microheterogeneity of nucleotide sequences in the DNA of these organisms .
' 8 Stable , hybrid partial diploids thus provide a convenient complementation system without significant possibility of recombination .
In this procedure F'his factors bearing appropriate E. coli hi VOL .
65 , 1970 mutations are introduced into the Salmonella his auxotroph .
Plating upon histidine-deficient media provides a sensitive test for even low levels of expression of the histidine biosynthetic enzymes .
The Salmonella his mutations used in these tests are included in Figure 1 .
Matings were conducted as detailed in Materials and Methods ; results appear in Table 2 .
As in conventional tests , complementation using an F'his + factor yielded growth within 24 hours .
Specific patterns of complementation are observed in tests using F'his - factors .
In many cases this complementation is at a relatively low level , being revealed upon 48-hour incubation .
Negative tests revealed no growth after incubation for six days .
Mutant his-3601 prevents expression of hisG , hisD , and hisC while clearly permitting expression of more distal genes in the presence of the appropriate episomal his-mutants .
Both hisOG203 and hisOG2232 are polar in that they will not utilize histidinol as a source of histidine .
Both fail to complement for hisD or hisC using episomes but do complement for expression of hisB and the other distal genes .
Likewise , all eight his OGD mutants fail to complement for F'hisC - but will permit expression of more distal genes .
From these data , the mutants cited show polar effects preventing expression of contiguous , intact genes while allowing complementation for certain sets of distal ones .
Comparable patterns are seen using hisOGDCB57 and hisOGDCBH2253 .
Each of these strains complements for expression of hisl and hisE but each fails to express one or more complete genes adjacent to the end of the deletion .
Alternative explanations for these findings have been considered .
typhimurium his + hisG hWD hiWsC hisB-HAFIE hisA hisF hisI hisE SC278 ( pro- ) + + -- + + + + + + + hisOG203 , arg - + + -- + + + + + + + hisOG2232 + + -- + + + + + + + hisOGD2369 + + -- + + + + + + + hisOGD646 + + -- + + + + + + + hisOGD22 .33 , leud + + + + hisOGD2225 + + + + + hisOGDCBS7 aro ' + + + + + hisOGDCB2226 + + + + + hisOGDCBH2253 + + ------- + + + hisOGDCBHAF-644 + + ------- + + + Response is indicated according to : + + , growth in 24 hr ; + , growth in 48 hr ; - , no growth in six-days incubation at 37 ° .
a F ' factors are his + for entire his operon except for the specific genes noted in each column .
b One each hisD mutant from the major complementing types was used ( Da , Db , Dab ) .
c Two separate hi8B mutants were used in parallel experiments .
d hisOGD2233 is representative of five mutants deleted for the same region within the his operon .
e Responds to Phe plus Tyr .
the mutants to be in fact totally polar , growth in these tests could occur if the F'his - mutant reverted on the plate , allowing expression of an intact F'his + operon , or if appropriate Salmonella-E .
coli recombinants were established among the sets of his-alleles in each cross .
In ruling out these possibilities , we obtained additional auxotrophic markers in several of the Salmonella strains to be used as recipient .
Clones subsequently scored as positive for complementation were then isolated and their genetic components verified by : ( a ) mating out the F'his - episome ; ( b ) curing the complemented strain of its episome by growing the strain in acridine orange and demonstrating the presence of the original Salmonella his-mutant ; and ( c ) testing for absence of conjugation by mating for transfer of markers ( trp + and metG + ) on either side of the his operon .
The possibility that the mutants are only highly polar was also considered .
Failure to detect complementation in this case might be theoretically due to intragenic negative complementation .
A limited production of the normal Salmonella gene polypeptide product , and an adequate production of mutant E. coli polypeptides from the corresponding gene could result in nonfunctional enzyme polymers .
This is not likely .
No evidence for such effects has appeared in Salmonella hisD-and E. coli F'hisD - mutant complementation tests : a survey involving 150 S. typhimurium mutants showed this type of intrageneric complementation to be essentially identical with the pattern found in abortive transduction tests involving only Salmonella .20 The results in Table 2 are equally negative using episomal hisD-mutants of either complementing ( Da or Db ) or noncomplementing type ( Dab ) .
Moreover , the hisG , hisC , and hisA genes have never shown evidence of intragenic complementation of any type .6 Failure to express these intact genes can not be due to the lack of normal translation initiators for these genes .
Mutant hisDC129 is a deletion which has lost a region extending to either side of the hisD-hisC boundary .
This mutant i transducible to wild type by his-3601 and hisOG203 .
Comparable transduction of hisOGDCB57 may be effected by using mutants deleting the distal portion of the his operon .
Presence of the normal hisD translation initiator in strains with his-3601 or hisOG203 may be deduced from the fact that both of these will revert to capacity to grow on histidinol as a source of histidine by events which map in hisG .21 ' 22 We conclude there are two natural initiators internal in the histidine operon which we designate P2 and P3 .
P2 is in hisC or at the hisC-hisB boundary , and P3 is in hisF or at the hisF-hisI boundary ( see Fig. 1 ) .
Previously published data from abortive transduction studies of hisOG203 and hisOGDCB57 gave different interpretations of the nature of polarity in these mutants .8 Detection of intergenic complementation is greatly increased in partial diploids using episomes , relative to the minute colonies formed in abortive transduction .
However , the scoring of abortive transductants itself has been facilitated more recently by the use of amino-acid supplements .6 We have examined strains SC278 and hisOG203 in reciprocal tests of abortive transductions using three D , three C , three B , one CB , two H , three I , and one each of F , A , and E his-mutants ; hisOGDCB57 was examined similarly using the H , A , F , I , and E mutants .
Our data from abortive transduction are all consistent with the patterns seen in episomal complementation .
The deletion mutations involving hisO which end in hisC or in hisF , ie. , his-OGDC63 , hisOGDC538 , hisOGDCBHAF644 , do complement for the genes directly following them in the operon order .
Although their properties are consistent with the evidence for P2 and P3 , lack of polar effects by these tests may arise from the effect of a promoter coupled by each deletion from outside the operon .
Existence of such promoter capacity has been suggested by others as a basis for expression of relatively nonpolar hisOG deletions .
' These latter mutants , of which hisOG1304 is shown in Figure 1 as an example , were obtained from OG203 by mutation to growth on histidinol as a source of histidine .
' Of the available deletion mutants ending distal to hisG , only hisOGDCB2226 is interpreted to be in a category analogous to hisOGi304 .
The degree of expression of distal genes due to the function of P2 or P3 has been considered .
From Table 2 this expression in merodiploids is typically sufficient for growth at least within 48 hours .
In the absence of the functional pathway , for example in hisOG203 without any F'his factor , P2 appears to be only minimally expressed .
Previous assays of extracts of hisOG203 for the hisB enzyme histidinol phosphate phosphatase failed to detect any activity ( less than 0.05 wild-type repressed level ' 5 ) .
Our assays of histidinol phosphatase confirm this observation with respect to both hisOG203 and to SC278 .
We did find qualitative evidence for the presence of the hisA enzyme , isomerase , however .
Using crude extracts of hisOG203 cells , isomerase was also undetected , but reproducible activity was found after partial purification ( protamine sulfate and ammonium sulfate fractionations similar to the published procedure `` 6 ) .
Extracts of his-HA 134 were treated identically as a control , and yielded no evidence of isomerase activity .
Assays of hisI or hisE as the result of P3 expression have not been attempted Properties of his-3601 : His-3601 is a revertable mutant which maps by transduction in the region covered by the deletion mutant hisOG203 .
Further mapping has taken advantage of the property of histidine-constitutive mutants to form wrinkled colonies on plates with high glucose .23 Mapping crosses are diagrammed in Figure 2 .
The method assumes a greater frequency of double cross-over events than quadruple events in this region .
This is supported by recovery of a higher proportion of wrinkled transductants when the more proximal hisG205 mutant is used as recipient relative to when hisG70 is used ( Table 3 ) .
By this method the relative mapping order is his-3601 , hisO1242 , hisG .
Mutant his-3601 has been more extensively mapped in this position by D. B. Fankhauser and P. E. Hartman using additional hisO constitutive mutants .24 From studies presented elsewhere mutant his-3601 does not appear to be suppressible .2 ' Induced initiator mutants : A class of point initiator mutants arising in hisOG203 has been described by Marie St. Pierre .22 These mutants , characterized by his-1306 , map in hisG and allow expression of hisD as detected by growth of the strains upon histidinol as a source of histidine .
When SC278 was plated upon histidinol revertants were isolated which were shown to be auxotrophic when tested upon minimal-medium .
These revertants are also due to mutations in hisG .
They comprise three separate categories as distinguished by their properties after subsequent reversion of the his-3601 mutation .
The functional level of these induced initiators has not been determined .
Numerous studies indicate the his operon to be a repressible system of the Jacob-Monod type .
Although operator-constitutive mutants are known , no mutations unique to the his promoter have been described .
His-3601 and extended deletion mutants of the hisOG203 type may owe their highly polar properties to a number of possibilities involving either transcription or translation .
Whether it represents a deficiency in transcription or translation , his-3601 maps proximal to some known operator constitutive mutants .
Thus , at least part of the his operator is indicated to lie distal to P1 The failure of expression of intact genes in his-3601 , hisOG203 , and similar deletions involving the proximal region is not due to the lack of potentially functional translation initiators .
Goldberger and colleagues have shown two modes of derepression of the his operon , sequential and simultaneous .3 Simultaneous derepression is interpreted as due to the attachment of ribosomes at the beginning of all genes in the his polycistronic mRNA .
Since we have shown the nonfunction of at least two genes proximal to P2 and P3 , we interpret P2 and P3 as being transcription initiators .
These initiators have not been detected in repression studies with a functional P1 present .
This is not surprising in the case of P2 which appears from enzyme assays to be expressed at only very low levels in the absence of P1 .
Moreover , expression of P1 may dominate over any secondary promoters ; there is suggestive evidence of this in the tryptophan operon.25-7 It is possible that the relatively high expression of the internal initiators as seen by growth in complementation tests is related to in-vivo enzyme stabilization in the presence of the other enzymes in the pathway .
It is also conceivable that the product of an additional element proximal to hisO accentuates expression of the internal initiators .
In our test this product would be provided by the complementing episome .
Fine structure mapping of P2 and P3 has not yet been attempted .
Should they map internally in the structural genes , P2 and P3 could represent initiators analogous to certain prototrophic mutations in the tryptophan operon .28 It is possible that they both map at gene boundaries , and may be vestiges of formerly efficient promoters in a preoperon stage of evolutionary development .
In this regard , the first internal promoter described , P2 in the S. typhimurium trp operon , does map at the boundary between structural genes .27 It is likely that this trp P2 is a transcription initiator in view of data obtained with the closely related E. coli trp operom .26 The use of nonrecombinant complementation in operon analysis offers a number of possible advantages .
Strains with hisOG203 have a normal D translation initiator .
Any mutationally derived initiators in hisD detectable when his-0G203 / F'hisC - reverts to grow on minimal-medium would thus be transcription initiators .
Similarly , partial diploids with hisOGDCB57 allow the search for transcription initiators arising in hisH and hisA .
Extension of the same methods may detect natural or mutationally derived intragenic translation initiators , or mutationally derived internal transcription terminators .
The latter should be detected were they to arise in the genes G , D , B , H , or A in the his operon .
Our thanks go to J. Roth for numerous strains , to P. E. Hartman for his generous assistance , and to R. F. Goldberger for a sample of the substrate for assays of hisA isomerase .
Since this work was completed , Dr. P. E. Hartman has indicated an additional mutant his-2321 to be a histidinol nonutilizing strain whose mutation maps under the region of hisOG203 .
It will recombine with two proximal hisG mutants ( hisG2101 and hisG200 ) but not with his-3601 .
We find his-3601 and his-2321 to be identical as regards their patterns of complementation with episome-borne mutants .
* This work was supported by grant GM 11079 from the U.S. Public Health Service .
t Present address : Department of Molecular Biology , University of Aarhus , 8000 Arhus , Denmark .
1 Ames , B. N. , R. F. Goldberger , P. E. Hartman , R. G. Martin , and J. R. Roth , in Regula tion of Nucleic Acids and Protein Biosynthesis , ed .
V. V. Koningsberger and L. Bosch ( New York : Elsevier Publishing Co. , 1967 ) , p. 272 .
2 Kovach , J. S. , M. A. Berberich , P. Venetianer , and R. F. Goldberger , J. Bacteriol. , 97 , 1283 ( 1969 ) .
3Berberich , M. A. , J. S. Kovach , and R. F. Goldberger , these PROCEEDINGS , 57 , 1857 ( 1967 ) .
4 Fink , G. R. , and R. G. Martin , J. Mol .
5 Venetianer , P. , M. A. Berberich , and R. F. Goldberger , Biochim .
6 Loper , J. C. , M. Grabner , R. C. Stahl , Z. Hartman , and P. E. Hartman , in Brookhaven Symposia in Biology , no. 17 ( 1964 ) , p. 15 .
7 Hartman , P. E. , J. C. Loper , and D. Serman , J. Gen. Microbiol. , 22 , 323 ( 1960 ) .
8 Hartman , P. E. , Z. Hartman , and D. Serman , J. Gen. Microbiol. , 22 , 354 ( 1960 ) .
9 Riyasaty , S. , and J. F. Atkins , J. Mol .
' ° Smith , H. O. , and M. Levine , Virology , 31 , 207 ( 1967 ) .
Fink , G. R. , T. Klopotowski , and B. N. Ames , J. Mol .
12 Hartman , P. E. , et al. , personal communication .
13 Berkowitz , D. , J. M. Hushon , H. J. Whitfield , Jr. , J. R. Roth , and B. N. Ames , J. Bacteriol. , 96 , 215 ( 1968 ) .
14 Roth , J. R. , personal communication .
' 5 Ames , B. N. , B. Garry , and L. R. Herzenberg , J. Gen. Microbiol. , 22 , 369 ( 1960 ) .
16 Margolies , M. N. , and R. F. Goldberger , J. Biol .
17 Perrin , D. , in Cold Spring Harbor Symposia on Quantitative Biology , vol .
`` Zinder , N. D. , Science , 131 , 813 ( 1960 ) .
`` Loper , J. C. , unpublished observations .
20 Atkins , J. F. , A. Marus , and J. C. Loper , Bacteriol .
21 Atkins , J. F. , Ph.D. thesis , Dublin University ( 1969 ) .
22 St. Pierre , M. L. , J. Mol .
23 Roth , J. R. , D. N. Anton , and P. E. Hartman , J. Mol .
24 Fankhauser , D. B. , and P. E. Hartman , personal communications ; see also Fankhauser , D. B. , Genetics , 61 , S17 ( 1969 ) .
25 Morse , D. E. , and C. Yanofsky , J. Mol .
27 Margolin , P. , and R. H. Bauerle , in Cold Spring Harbor Symposia on Quantitative Biology , vol .
28 Wuesthoff , G. , and R. H. Bauerle , Bacteriol .