A SALMONELLA TYPHIMURIUM LOCUS INVOLVED IN THE

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1 A SALMONELLA TYPHIMURIUM LOCUS INVOLVED IN THE REGULATION OF ISOLEUCINE, VALINE AND LEUCINE BIOSYNTHESIS1 RENEE R. ALEXANDER AND J. M. CALVO Department of Biochemistry and Molecular Biology, Cornell Uniuersity, Ithuca, New York Received August 19, 1968 HE genes determining the enzymes of isoleucine and valine biosynthesis in Escherichia coli and Salmonella typhimurium are clustered in one region of the genome near mte and in E. coli are organized into two or three operons (RAMAKRISHNAN and ADELBERG 1965a, 1965b). The expression of these operons is under the control of isoleucine, valine and leucine (FREUNDLICH, BURNS and UMBARGER 1962). Leucine biosynthesis in Salmonella is effekted by four enzymes, the structures of three of which are determined by four adjacent cistrons (BURNS, UMBARGER and GROSS 1963; MARGOLIN 1963). The expression of these genes in terms of enzyme levels is governed by the intracellular concentration of leucine and requires the functioning of several genetic elements. This conclusion was reached from studies on the coordinate synthesis of leucine enzymes (BURNS et d. 1966) and from an analysis of mutants isolated by resistance to a leucine analog, 5,5,5 -trifluoro-~~-leucine ( RENNERT and ANKER 1963; CALVO, FREUNDLICH and UMBARGER 1969). Many of the strains which are resistant to this analog are unable to regulate leucine production and as a result, overproduce leucine. One class of such mutants has high, constitutive leucine enzyme levels and the mutant sites of these strains lie in a region adjacent to the leucine structural cistrons. A second class has constitutive levels of enzymes involved in leucine, valine and isoleucine biosynthesis and the mutant sites involved are unlinked by transduction to the leucine operon (CALVO et al. 1969). The purpose of this communication is to identify a locus for the latter class, fir, which is located between pure and gal on the Salmonella genome. MATERIALS AND METHODS Bacterial strains: The strains of Salmonella typhimurium used in this study, all derivatives of strain LT2, are described in Table 1. The nomenclature used follows the standards proposed by DEMEREC et d. (1966). Media. Nutrient broth (Difco, 8 g; NaC1, 4g; H,O, 1 liter) and nutrient agar (Baltimore Biological Laboratory, 23g; NaC1, 4g; H,O, 1 liter) were used as complete media. A minimal salts solution (SSA) contained per liter of distilled water: K,HPO,, 10.5 g; KH,PO,, 4.5 g; (NH,),SO,, 1.0 g; sodium citrate dihydrate, 0.97 g; MgSO,, 0.05 g. SSA supplemented with 0.2% glucose or 0.2% glucose and 1.5% agar served as liquid and solid minimal media, respec- Supported by National Science Foundation Grant GB Genetics 61 : March 1969.

2 cn P C F > M x tr M w % U 4 TABLE 1 Characteristics of S. typhimurium strains used in this study Strain" Genotype* Remarks or phenotype Source or parent Hfr Strains Order of injection: S414 his-23 gal-50 0-pdx-ilu-thr-trp-aroB K. SANDERSON CVl00 his-23 gal-50 0-pdx-ilv-thr-trp-aroB S414 flr- 100 SU576 purc7 stra 0-pdx-ilu-thr-trp-aroB H. SMITH F- Strains ilua212 K. SANDERSON iluc8 K. SANDERSON metb81 K. SANDERSON thra8 K. SANDERSON pan-2 K. SANDERSON proai07 K. SANDERSON pure8 K. SANDERSON asc-i K. SANDERSON S268 meta22 trpe2 M. DMEREC glt-3 tyr-40 nica13 Map position (SANDERSON 1967) 120 (origin) (origin) r 120 (origin) ci grows on either gluta- K. SANDERSON 36 mate or proline 12? (for nica13)

3 CONTROL LOCUS FOR LEU, VAL AND ILE 541..

4 542 R. R. ALEXANDER AND J. M. CALVO tively (M medium, M plates). Enriched minimal agar contained 0.01 (EM) or 0.02 (2 EM) percent (w/v) dehydrated nutrient broth. Plates for the assay of leucine excretion (SL plates) were prepared as follows: Twenty ml of solid M medium were poured and allowed to solidify in Petri dishes. A 10 ml portion of the same agar, cooled to 47 C and containing 4 X IO8 cells of strain leu BCD 39 per ml was then layered on top. Maintenance of Hfr strains: Strain SU576 is a stable Hfr strain. S414, on the other hand, reverted to F+ and periodically, effective males were reisolated by the following procedure. Twelve single colonies of S414 were picked with sterile tooth picks and smeared on a nutrient agar plate in patches about 1.5 cm in diameter. Each colony was also spotted on a separate nutrient agar plate so that promising isolates could be retrieved from the master plate after the patch test. After incubation for 24 hr, these patches were replicated on to a recipient lawn of cells prepared by spreading 0.1 ml of an overnight broth culture of a suitable recipient (usually pure8) on an EM plate. Patches showing heavy growth as a result of recombination were scored and the effective Hfr colonies were picked from the master plate. Uninterrupted mating experiments: In these experiments, donor and recipient bacteria were simply mixed and allowed to mate on the surface of a minimal agar plate. Overnight broth cultures (about 4 x 109 bacteria/ml) were centrifuged, washed, and resuspended in an equal volume of SSA solution. The donor suspension was diluted by serial tenfold dilution and 1 ml portions of the 10-1, 10-2 and 10-3 dilutions were added to 1 ml portions of the undiluted recipient cells. 0.1 ml of the mixture was plated on minimal agar plates supplemented so as to enable selection for a given marker. In some experiments, the number of recombinants was increased by adding 0.1 ml of nutrient broth to each plate. Samples of the donor and recipient plated separately served as controls. Recombinants were scored after incubation for 48 hr for incorporation of unselected markers. Interrupted mating experiments: The procedure used has been described in detail by SANDER- SON and DEMEFLEC (1965). When selection was made for nic+, this procedure required modification because the small amount of broth included in the plating contained sufficient growth factor to give a high background lawn which interfered with the results. The following procedure was adopted for these crosses: After vortexing to interrupt mating, the sample of mating mixture was passed through a 25 mm Millipore filter (type HAWP, Matheson-Higgins Co. Inc., Cambridge, Mass.) and the cells were resuspended in 2 ml of SSA and diluted appropriately prior to plating. A commercial preparation of flagellar antiserum was used in several of the interrupted mating experiments to reduce the number of plate matings (manuscript in preparation). Difco Salmonella flagellar antiserum H (mixed group i and 1 complex) was added to the plating soft agar to give a final dilution of 1:3200, together with sodium thioglycolate at a final concentration of 5 x 10-'~. The latter was added to inactivate the merthiolate added by the manfacturer as a preservative. Incubation procedures: All cultures were incubated at 37 C. Liquid cultures were aerated by bubbling filtered air through a tube containing 10 ml of medium or by placing cultures in Erlenmeyer flasks on a rotary shaker. Transduction techniques: Transductions were mediated by PLT22 type HI phage using the procedure described by MARGOLIN (1963). Scoring of unselected markers: All of the fluoroleucine-resistant strains used in this study overproduce and excrete leucine. This latter property was routinely scored by an auxanographic test ( CALVO et al. 1969). The ability to utilize galactose as ssle carbon source was determined by transferring recombinants to M plates lacking citrate and containing 0.2% galactose in place of glucose and scoring growth as plus or minus after incubation for 24 hr. Auxotrophic phenotypes were determined by transferring recombinants to M plates and to M plates supplemented with growth factor and scoring growth as plus or minus after 24 hr. RESULTS Approximate location of the flr locus: CVlOO was selected as a fluoroleucineresistant mutant of Hfr strain S414. This strain over-produced and excreted leu-

5 CONTROL LOCUS FOR LEU, VAL AND ILE 543 cine as judged by auxanographic tests and the mutant site responsible for this phenotype was not linked to the leucine operon by transduction. The specific activities of some of the enzymes involved in isoleucine, valine and leucine biosynthesis were moderately elevated in CVlOO in comparison with the parent strain but lower than in some other strains isolated by the same method (CALVO et al. 1969). The gradient of transmission of markers in conjugation (JACOB and WOLLMAN 1961) proved to be a suitable method for the initial location of the flr locus. This method is based upon the fact that the frequency of recombinants decreases with increasing distance between the selected marker and the origin of the Hfr strain, presumably because there is a constant chance with time that a mating pair will break apart. Hfr strain CVIOO was used in conjugation experiments with 13 different auxotrophic derivatives of the F- strain LT2. These recipients, kindly supplied by DR. K. SANDERSON, were chosen because their mutant sites are widely scattered around the chromosome (SANDERSON and DEMEREC 1965). In each cross, prototrophic recombinants were selected and these were scored for the donor unselected characters, leucine excretion and galactose utilization. For this type of analysis, the expected results are as follows: a) if the unselected marker lies between the origin of the Hfr and the selected marker, roughly 50% of the recombinants may be expected to have the donor unselected marker (HAYES 1964) j b) if the unselected marker lies far distal to the selected marker, less than 50% of the recombinants will inherit the donor unselected marker because of the gradient of transmission effect (JACOB and WOLLMAN 1961) ; c) if the unselected marker is close to the selected marker, more than 50% of the recombinants will inherit the donor unselected marker. For conjugation in Escherichia coli, such linkage between the selected and unselected marker is observed when the two are within 3 min of each other (HAYES 1964). These three possibilities are diagrammatically illustrated in Figure 1. The results of these crosses are shown in Figure 2. Originally, the unselected donor gal marker was expected to serve as a control for the method. Unexpectedly, it proved to be an ideal control because its appearance among the recombinants paralleled that seen for the flr locus. The incorporation of ji r and gal from the donor was very low when selection was macle for early markers (metb+, pan+), Origin,U S+ c- S- c+ Donor Recipient / s+ U c- \ S- C+ FIGURE 1.-Relationship between selected and unselected markers in conjugation experiments. S, selected marker; U, unselected marker; C, donor contra-selective marker. See text for explanation.

6 544 R. R. ALEXANDER AND J. M. CALVO began to rise with selection for proa+ and pure+, and was significantly greater than 50% with selection for glt+. For markers distal to glt, the percentage incorporation of the two markers dropped abruptly. We conclude that the flr locus is close to both gal and glt on the chromosome. To confirm the proximity between gal and flr, CV125 (an independently isolated fluoroleucine-resistant derivative of ara-9 gal-205 ) was used as a recipient in a cross with donor SU576, selection being made for galactose utilization. In this cross, 80% of the recombinants acquired the donor flr marker. (Figure 2, see value for gal). It is not known if the flr mutations in CV125 and CVlOO are allelic. Expectations b) and c) above were realized in this study but not a). In Figure 2 it is seen that fewer than 50% of the recombinants inherited the donor unselected markers when late markers were selected (e.g. trpd+, metgf). In other experiments where selection was made for late markers the percentage of unselected donor character in the progeny approached 50%. In still other experiments in which the same cross was repeated many times the percentage was found to vary from 46 to 85%. The reasons for these anomalies are not known. Interrupted mating experiments: A. The order pure-flr-gal. The times of entry of pure8, flr-100 and gal-50 were determined in interrupted mating experiments. pure served as the selected marker and prototrophic recombinants were analyzed for flr and gal unselected markers (Figure 3). The data for the times of entry for these three genes obtained from four interrupted mating experiments are sum- met8 ora-9 pma asc-1 glt-3 pyrd tryd I I. I I, I. I. I I I ' I ' I' I I.I Nv thra pan-2 purenk-i3 gal pyrc m:tg I20 I30 lje Ib Map position of selected marker (minutes) FIGURE 2.-Analysis of recombinants from uninterrupted mating experiments for unselected donor markers. Cross: donor CVlOO his-23 gal-50 fir-100 x recipients identified above abscissa. The crosshatched bar represents percentage of donor fir+ among gal+ recombinants from the cross: donor SU576 purc7 x recipient CVl25 ara-9 gal-205 fir-125. The map positions listed are from SANDERSON and DEMEREC (1965).

7 CONTROL LOCUS FOR LEU, VAL AND ILE ] Time of interruption (minutes) FIGURE 3.-Time of entry for pure8, gal-50 and flr-64. Donor CVlOO his-23 gal-50 f7r-100 X recipient pure8. In this and subsequent time of entry figures, the data have been corrected for background plate matings by subtracting the average number of recombinants from two or three samples interrupted before the time of entry of the selected markers. This background level never exceeded 10% of the maximum recombinants observed at much later times of interruption. 0-0, total prototrophic recombinants; A-A, prototrophic recombinants having donor f7r marker; e-, Prototrophic recombinants having donor g d marker. marized in Table 2. In Figure 3 and subsequent figures showing the results of interrupted mating experiments, the curves have been corrected for background plate matings. The number of such plate matings never represented more than 10% of the maximum number of recombinants observed in an experiment. To convert times of entry to map positions, 25 was subtracted from the former (18 min from the origin to zero on the map and 7 min to account for a delay period prior to the initiation of transfer (SANDERSON and DEMEREC 1965) ). The average values of four time of entry determinations place the f;r locus two min to the right of pure and four min to the left of gal (Table 2). B. The order Origin-gal-glt. CV352, a derivative of get-3 unable to utilize galactose as a carbon source was isolated from glt-3 following treatment with N-methyl- N'-nitro-N-nitrosoguanidine ( Aldrich Chemical Co., Milwaukee) using the procedure of ADELBERG, MANDEL and CHEN (1965). This strain was used to determine the relative order of gal and glt using SU576 as donor. Samples from an interrupted mating experiment were plated on minimal medium to select for glt+ recombinants and on minimal medium containing galactose as sole carbon source and glutamic acid to select for gal+ recombinants (Figure 4 and Table 2). The times of entry for gal and glt, 46 and 55 min respectively, indicate the order,

8 ~~ 546 R. R. ALEXANDER AND J. M. CALVO TABLE 2 Time of entry of markers located in the pure-pyrd region Time of entry Cross Selection Unselected Value Map position Donor X Recipient made for marker anal) zed (min) Ai eraye (InlnJ CVlOO his-23 gal-50 puref 39,40 1 $o flr-100 x pure8 39,41 SU576 purc7 x CV352 glt-3 gal-302 S414 his-23 gal-50 x CV325 glt-3 flr-325 gal+ glt+ gzt+ SU576 purc7 x nic + S268 nica 13 CVlOO his-23 gal-50 X S268 nica13 SU576 purc7 x pyrd 121 nict flr- 100 gal-50 glt-3 pure, 15 41*743*1 42 flr, 17 43*, 43'1 45*, 46' gal, , glt, 29 nic, PYrD, 33 * Time of entry determination for an unselected marker. Origin-gal-glt. Analysis of the gal+ recombinants for glt (in a manner analogous to that shown in Figure 6) gave another estimate of 54 min for the time of entry of g2t. In addition, two separate estimations for glt were obtained from data from the cross S414 x CV325 in which selection was made for glt+ (Table 2). The order Origin-gal-glt was confirmed by analyzing the recombinants from the gal+ selection for the unselected glt character and vice versa (Tab!e 3). Analysis of glt+ recombinants for the donor gal+ marker gave approximately constant values around 50% at all sampling times whereas glt+ colonies among the gal+ recombinants increased in number with time. This evidence unequivocally points to the order: Origin-gal-glt. C. The order Origin-gal-nic. The results of conjugation experiments in which nica13 was used as a selected marker did not always support unambiguous conclusions due to heavy background growth of the recipient. Using the technique described in METHODS, it was possible to obtain a reasonably linear increase in recombinants with time. Linear regression analyses of the data obtained from two interrupted mating experiments place the time of entry between 47 and 50 min (Figure 5a and Table 2). This value is somewhat larger than the time of entry of gal (46 min) j however, because of the uncertainty in the time of entry value for nic, a firm conclusion concerning their order is not warranted from these data. Data which further support the order Origin-flr-gal-nic are shown in Table 4;

9 CONTROL LOCUS FOR LEU, VAL AND ILE 547 Time of interruption (minutes) FIGURE 4.-Time of entry for gal and glt. Donor SU576 purc7 x recipient CV352 gli-3 gal , gal+ recombinants (linear regression analysis, Y = -94,225 -k 2007X) ; 0-, glt+ recombinants (Y = -80, X). nic+ recombinants from the cross CVlOO x S268 were analyzed for the unselected donor markers, gal and flr. A constant value for the percentage of unselected donor character among the recombinants was observed at all times of interruption (average of 76% for gal and 42% for flr). This implies that when selection is made for nic+, both gal and flr have already entered and that gal is closer to nic than flr. TABLE 3 Analysis of glt+ and gal+ recombinants for corresponding unselected markers Cross: donor SU576 purc7 x CV352 glt-3 gal-302 Selection for glt+ Selection for gal+ gal+ glt+ * glt+ gal+ t Time of interruption ~- Percent Percent of mating (min) glt+ gal / /2oo 1 92/ / / / / / / / * The number of glt+ recombinants having gal+ divided by the total number of recombinants. j- The number of gal+ recombinants having gzt+ divided by the total number of recombinants.

10 548 R. R. ALEXANDER AND J. M. CALVO 200, ,000- I20,OOO- I /O I d 00,000 / IO0.-c 90 FIGURE 5.-Time Time of interruption (minutes) of entry for nica13 and pyrd (a) Donor SU576 purc7 x recipient S268 nica13 (Y = -186, X), (b) Donor SU576 purc7 x recipient pyrd121. D. Time of entry of pyrd. The time of entry for pyrd from an interrupted mating experiment was 58 min which locates this marker at 33 min on the map (Figure 5b). Determination of linkage between uarious markers in the pure-pyrd region by transduction: On the basis of 1) times of entry and 2) high frequency of joint inheritance in conjugation experiments, several markers in the pure-pyrd region seemed sufficiently close to warrant a transduction linkage test (Table 5). The mutant site of strain CV112, considered to be an operator constitutive site (CALVO et al. 1969), showed linkage as expected to leu but not to pure, glt, or pyrd and thus served as a control for the method of scoring the leucine excretion phenotype. Three fluoroleucine-resistant strains, CV100, CV125 and CV325, were tested TABLE 4 Analysis of nic+ recombinants for incorporation of unselected donor markers gal-50 and flr-100 Cross: donor CVl00 his-23 gal-50 flr-io0 x S268 nica13 nic+ flr-ioo* nic+ galdot Time of interruption of mating (min) nic+ Percent nic+ Percent 45 22/ / / / / / / / * The number of nicf recombinants having flr-100 divided by the total number of recombinants. t The number of nic+ recombinants having gal-50 divided by the total number of recombinants.

11 CONTROL MCUS FOR LEU, VAL AND ILE 549 TABLE 5 Determination of linkage between various markers in the pure-pyrd region by transduction Number of Number of transductants Donor phage Unselected transductants having donor grown on: Recipient* character scored analyzed unselected marker CV112 leu02004-f leubcd39 leu (98%) ara-9 gal-205 If pure8 leu n glt-3 leu02004-f I, pyrdl2l leu02004-f CV125 ara-9 leubcd39 flr gal-205 flr-125,i pure8 Pr R git-3 flr r, pyrdl2l flr 250 0, urd: flr DR14cS P. 30 0, S268 nica13 gal 98 0,, glt-3 gal CV325 glt-3 gal-205 Pr flr-325, n S268 nica13 fir S268 nical3 glt CVlOO his-23 S268 nica13 flr gal-50 flr- 100 n glt-3 flr rr asc-i flr f# glt-3 gal f> S268 nica13 gal S268 nica 1 3 glt-3 nic glt-3 I, CVlOO gal-50 CV125 gal-205 glt glt * In each cross, selection was made for the wild type allele of the recipient marker shown. +leu02004 is an operator constitutive mutation and like the flr marker, was scored by an auxanographic test for leucine excretion. $ DR14c is unable to utilize 2- deoxyribose as a sole carbon source. When used as a recipient, this strain gave very low numbers of transductants. by transduction for linkage of their mutant sites to loci in the pure-pyrd region. No co-transduction was observed between these markers and pure, glt-3, pyrd, arog (WALLACE and PITTARD 1967), gal-205, nica13, asc-i, and a locus involved in the metabolism of 2-deoxyribose which was recently reported to be in this region (HOFFEE 1967). In addition, no linkage was found between nica13 and gal, between nical3 and glt, or between glt and gal. Search for other flr loci: A major goal of this work was the identification of all loci involved in the control of leucine, isoleucine and valine biosynthesis. The flr-100 and flr-125 sites map in the pure-pyrd region of the genome. A group of

12 550 R. R. ALEXANDER AND J. M. CALVO TABLE 6 Analysis of gal+ and araf recombinants for donor unselected flr+ marker Cross: donor SU576 purc7 x recipients (fluoroleucine-resistant deriuan ues of ara-9 gal-205) Selection for: am+ am+ flr+ X 10O/ura+* gal+ flr+x IO!l/gu?* Map Linkage to Conjugation Con] uganon region leucine operon Plate interrupted Plate interrupted suggested by transduction Recipient mating at 40 min matlng at 60 min by data analysis cv112 CV114 CV115 CV116 CV117 cv122 CV123 CV124 CV125 CV126 CV (14)t * The number of recombinants having fir+ divided by the total number of recombinants. One hundred recombinants from each cross were analyzed. I. Values in parentheses represent a second determination. fluoroleucine-resistant derivatives of ara-9 gal-205 were examined for linkage of their flr sites to the pure-pyrd region. These strains (CV112-CV127) were first used as recipients in uninterrupted mating crosses with donor SU576, selection being made for ara+ and, in independent platings, for gal+ and the recombinants were analyzed for leucine excretion (Columns 2 and 4, Table 6). Strains CV112, CV126 and CV127, known from transduction studies to have loci conveying fluoroleucine resistance linked to the leucine operon, all showed very high joint incorporation of the leucine excretion characteristic and ara+. Joint inheritance of gal+ and the leucine excretion character seemed reasonable in two cases (36 and 42%) but unusually high in another (68%). The reason for this discrepancy is not known. The remaining eight strains analyzed showed joint inheritance of gal+ and the leucine excretion character ranging from 62% to 88%, suggesting that the flr loci in these strains are close to gal on the genome. This conclusion was not entirely justified by the results of the ma+ selection; 20-62% of the ara+ recombinants had the donor flr marker (Column 2, Table 6). From the results shown in Figure 2, one would have predicted that selection for any early marker such as am+ would have resulted in a low (about 5 % ) incorporation of a marker near gal because of a gradient of transmission. However, a different Hfr strain was used as donor in the two series of crosses (the donor strain used in the experiment described in Figure 2 was derived from S414) and it seemed possible that the frequency of breakage between mating pairs was lower in SU576 than it was in S414 resulting in greater frequencies of incorporation of late markers. This possibility was tested by analyzing ara+ recombinants for the incorporation of donor flr and gal markers from the crosses SU576 x CV125 and S414 x CV125. gal+ Yes no no no no no no no no Yes Yes

13 CONTROL LOCUS FOR LEU, VAL AND ILE TABLE 7 Gradient of transmission pattern obtained with two different Hfr strains 55 1 Cross Donor X Recipient SU576 purc7 x CV125 ara-9 gal-205 flr-125 SU576 purc7 x CV125 ara-9 gal-205 fir-125 I Mating procedure Plate mating 55/150 (37%) 66/150 (44%) Mating on filter followed by agitation 40/150 (27%) 86/150 (58%) in broth. S414 his-23 gal-50 x CV125 ara-9 gal-205 flr-125 Plate mating 5/150 (3.5%) I * Number of am+ recombinants having donor unselected marker divided by the total number of araf recombinants. The latter cross, however, could not be analyzed for gal because both parents were unable to utilize galactose. The data shown in Table 7 demonstrate that, indeed, SU576 and S414 transmit unselected late markers at different frequencies. The value of 3.5% flrf colonies among ara+ recombinants from the S414 x CV125 cross was expected on the basis of the data shown in Figure 2. The SU576 x CV125 cross showed a much higher incorporation of unselected late markers: fir+, 37%; gal+, 44.x. Possibly, SU576 gave a more shallow gradient than S414 because it is non-motile (unpublished data). The SU576 x CV125 cross was repeated under conditions more likely to produce a gradient. Donor and recipient cells mated on a Millipore filter were washed gently into broth and after varying periods of incubation with gentle shaking, were plated in soft agar without interruption. It was anticipated that gentle shaking in liquid would elicit a steeper gradient than mating on the surface of agar. This was not the case (line 2, Table 7) ; essentially the same results were obtained by either mating procedure. That a steeper gradient of transmission could be generated is seen from the results of an interrupted mating experiment with SU576 (Figure 6). When the mating was interrupted between 30 and 40 min, no donor gal or flr markers were seen among the araf recombinants. The times of entry of flr-125 and gal-205 were very close to those of flr-100 and gal-50, respectively (Table 2). The results above suggested that other ara-9 gal-205 fzr isolates might be checked for linkage of flr to the pure-pyrd region if a steeper gradient were established by artificial interruption. A high incorporation of donor flr+ among gal+ recombinants would be taken to mean that flr is located close to gal or between the origin and gal. A low incorporation of donor flr+ among ara+ recombinants with interruption at 40 min would signify that flr enters later than 40 min from the initiation of transfer. If the results conform within the above limits, we consider the map position of the flr marker to be within 5 min on either side of gal. The results of such experiments are seen in columns 3 and 5 of Table 6 and indicate that the flr loci of 6 strains, CV115, 116, 117, 122, 124, and 125 all map

14 552 R. R. ALEXANDER AND J. M. CALVO e 3 c.- X E 0.- c t E 800, , c 0 c a E 0 0 e r 0 400, , Time of interruption (minutes) FIGURE 6.-Time of entry for ara-9 gal-205 flr-125. Donor SU576 pus7 x recipient CV125 ara-9 gal-205 fir-125. Selection was made for ma+ recombinants. 0-0, araf recombinants; A-A, ara+ recombinants having donor fir+ marker; *-a, ara+ recombinants having donor gal+ marker. in the pure-pyrd region of the genome. In these experiments, interruption at 40 min did not completely sever linkage between ara and fzr (column 3, Table 6). The reason for this is not clear. DISCUSSION The gradient of transmission technique was very useful for initially determining the approximate location of the flr locus. However, the comparative study of SU576 and S414 given in Table 7 points out the importance of choosing the right donor for the study. The difference in behavior of the two strains may be explained by the fact that SU576 but not S414 is non-motile. The chance of pulling apart the mating pairs could be increased by the rapid motion of the motile strain s flagella. This would explain both the shallow gradient of transmission of SU576 and the observation that SU576 gives from 10 to 100 times higher numbers of recombinants than does S414. The data concerning the order of markers in the pure-pyrd region from this and the other studies are summarized in Table 8. It seems clear that the order pure-gal-pyrd is the same in Escherichia and Salmonella but there is some question concerning the relative distances between the three loci. The study of SANDERSON and DEMEREC (1965) suggested that the two genera differed markedly

15 CONTROL LOCUS FOR LEU, VAL AND ILE TABLE 8 Summary of mapping data 553 Order of markers as determined by: This study Time of entry for Time of entry for Analysis of unselected Escherichia, TAYLOR Salmonella SANQER~ON Time of Recombination markers from and THOMAN, and DW&C, entry2 frequency interrupted matingsf pure, 11 pure, asc-1, nica13,22... glt-3, 36 gal, 16 gal, PY~D, 20 Pya 41 pure, 15 Pr, gal, 21 nica13,23 glt-3, 29 Pya 33 pure fzr _..... gal glt gal (glt-3, nica13) nical3... mc PYrD... * Frequency of incorporation of unselected markers; data from Figures 3 and 8 were used in the determination of order. + Order was determined from data in Tables 3 and 4. The values represent map distances in minutes. in this region, the pure-pyrd distance being 13 minutes longer in Salmonella (22 versus 9 min). Our work indicates a shorter pure-pyrd interval, though it is still considerably longer than the corresponding region in Escherichia (1 8 versus 9 min). A comparison of the pure-gal interval from the three studies summarized in Table 8 points up a discrepancy. The interval of 6 min found in this study is similar to that found in E. coli (5 min) but is much smaller than that found by SANDERSON and DEMEREC (1965) (19 min). The reason for the difference between our data and those of SANDERSON and DEMEREC is not clear; the same methods and strains were used in both studies. The order pure-fir-gal is reasonably certain. The order pure-gal- (nica13, glt-3), although in conflict with data from SANDERSON and DEMEREC (1965), seems certain. What gives this conclusion some weight are the data from Tables 3 and 4. The proportion of donor glt+ markers among gal+ recombinants increases with increased time of contact between the parents and this fact cannot be readily explained on he basis of the reverse order. Similarly, the constant incorporation of donor gal+ among nic+ recombinants at different times of interruption argues, although less strongly, that nic is distal to gal. It is clear from Figure 2 that there is a meaningful correlation between the positions of the selected and unselected markers (relative to the origin) and the percentage of recombinants having the donor unselected marker. Thus, for the three types of arrangements shown in Figure 1, the percentage of recombinants having the donor unselected marker was, respectively, intermediate (right side of graph in Figure 2), low (left side of graph), and high (peak of graph). If recombination in these crosses was not affected by the nature 01 the individual markers, then the tips of the bars in Figure 2 might be expected to lie on two smooth curves, one for fir incorporation and one for gal incorporation. Several bar tips, particu-

16 554 R. R. ALEXANDER AND J. M. CALVO a L '""1 120 I IO 20 io Map position of selected marker (minutes) FIGURE 7.-Redrawing of Figure 2. The map positions for pure, gal, and pyrd are taken from Table 2; the remainder are from SANDERSON and DEMEREC (1965). glt, nic and usc have been positioned so that smooth curves for the incorporation of unselected markers could be drawn. Dashed line connects white bars (donor flr incorporation); dotted line connects black bars (donor gal incorporation). Crosshatched bar: see legend to Figure 2. larly those for asc, nic, and glt, do not lie on a smooth curve. In Figure 7, Figure 2 is redrawn using our time of entry data for pure, gal and pyrd (Table 2) and positioning glt, nic and ax so that smooth curves for the incorporation of unselected markers could be drawn. Assuming a smooth curve relationship is valid, estimates for the position of glt, nic, and asc from Figure 7 are 22,25 and 29 min, respectively. If the curves in Figure 7 are accurate, then linkage between selected and unselected markers greater than 50% occurs when the two markers are within 4 minutes of each other. A comparison of columns 2,3 and 4 of Table 8 points out that the recombination frequency and time of entry methods suggest different relative orders for glt-nic and asc-gal. We see no strong reason to favor the results of either method. The results of the co-transduction analyses are consistent with our conjugation data which revealed no two markers in the pure-pyrd region closer than 2 minutes. Since P22 phage transmits a length of chromosome of about 1 minute, linkage between any of the markers examined and fzr was not expected. This was unfortunate since linkage to some other marker by transduction would have facilitated the search for other leucine regulatory sites. Genetic loci known to be involved in the regulation of leucine production now number three: an operator locus defined by operator constitutive (CALVO et al. 1969) and operator negative (MARGOLIN 1963) mutants; a structural gene which codes for an endproduct inhibitable enzyme (CALVO et al. 1969) ; and the fzr locus

17 CONTROL LOCUS FOR LEU, VAL AND ILE 555 described here. The mutant sites of two strains of independent origin, flr-100, and flr-125, were found in this study to map close to gal and to have similar times of entry; their mutant sites may be allelic. In addition, the flr loci of six other strains were shown to map within 5 minutes of gal (Table 6). Thus, there is one and possibly several leucine regulatory site(s) in the gal region. An analysis of 50 fluoroleucine-resistant derivatives of glt-3 of independent origin has not brought to light any additional leucine regulatory loci (unpublished data). The biochemical consequences of a mutation in flr are not known in detail. Besides operator constitutive and feedback negative mutants, regulatory mutants are known which have altered regulatory proteins (GILBERT and MULLER-HILL 1966; PTASHNE 1967), altered amino acyl-trna synthetases (NEIDHARDT 1966) and still others with altered trna patterns ( SILBERT, FINK and AMES 1966). The flr locus described could conceivably code for any of the three mentioned above. The authors wish to acknowledge the help and advice of DR. K. SANDERSON. SUMMARY Mutants of Salmonella typhimurium have been isolated which have altered levels of enzymes involved in isoleucine, valine and leucine biosynthesis. The mutant sites of two such mutants, flr-100 and flr-125, map between pure and gal, 2 min from the former and 4 min from the latter. The mutant sites of six other strains map within 5 min of gal and may or may not be allelic with flr-100 and flr-125. The order of loci in the pure-pyrd region is pure-flr-gal-(nic, g1t)- pyrd. Estimates of the pure-pyrd and pure-gal distances from this work are somewhat lower than previous estimates, the former approaching the corresponding values found in Escherichia coli. LITERATURE CITED ADELBERG, E. A., M. MANDEL, and G. C. CHEN, 1965 Optimal conditions for mutagenesis by N-methyl-N -nitro-n-nitroso guanidine in E. coli K-12. Biochem. Biophys. Res. Commun. 18: BURNS, R. O., J. M. CALVO, P. MARGOLIN, and H. E. UMBARGER, 1966 Expression of the leucine operon. J. Bacteriol. 91 : BURNS, R. O., H. E. UMBARGER, and S. R. GROSS, 1963 The biosynthesis of leucine The conversion of or-hydroxy-p-carboxy-isocaproate to or-ketoisocaproate. Biochemistry 2 : CALVO, J. M., M. FREUNDLICH, and H. E. UMBARGER, 1969 Regulation of branched-chain amino acid biosynthesis in Salmonella typhimurium. J. Bacteriol. 97: DEMEREC, M., E. A. ADELBERG, A. J. CLARK, and P. E. HARTMAN, 1966 A proposal for a uniform nomenclature in bacterial genetics. Genetics 54: FREUNDLICH, M., R. 0. BURNS, and H. E. UMBARGER, 1962 Control of isoleucine, valine, and leucine biosynthesis. I. Multivalent repression. Proc. Natl. Acad. Sci. US. 48: GILBERT, W., and B. MULLER-HILL, 1967 Isolation of the lac repressor. Proc. Natl. Acad. Sci. U.S. 56: HAYES, W., 1964 The Genetics of Bacteria and their Viruses. Wiley; N.Y., pp ,

18 556 R. R. ALEXANDER AND J. M. CALVO HOFFEE, P., 1967 The genetic locus of deoxyribose metabolism in Salmonella typhimurium. Federation Proc JACOB, F., and E. WOLLMAN, 1961 Sexuality and the Genetics of Bacteria. Academic Press; N.Y., pp MARGOLIN, P., 1963 Genetic fine structure of the leucine operon in Salmonella. Genetics 48: NEIDHARDT, F. C., 1966 Roles of amino acid activating enzymes in cellular physiology. Bacteriol. Rev. 30: PTASHNE, M., 1966 Isolation of the h phage repressor. Proc. Natl. Acad. Sci. U.S. 57: RAMAKRISHNAN, T., and E. A. ADELFJERG, 1965a Regulatory mechanisms in the biosynthesis of isoleucine and valine. 11. Identification of two operator genes. J. Bacteriol. 89 : b Regulatory mechanisms in the biosynthesis of isoleucine and valine 111. Map order of the structural genes and operator genes. J. Bacteriol. 89: RENNFXT, 0. M., and H. S. ANKER, 1963 On the incorporation of 5'5'5'-trifluoroleucine into proteins of E. coli. Biochemistry 2: SANDERSON, K., and M. DENIFXEC, 1965 The linkage map of Salmonella typhimurium. Genetics 51: SANDERSON, K., 1967 Revised linkage map of Salmonella typhimurium. Bacteriol. Rev. 31 : SILBERT, D. F., G. R. FINK, and B. N. AMES, 1966 Histidine regulatory mutants in Salmonella typhimurium A class of regulatory mutants deficient in trna for histidine. J. Mol. Biol. 22 : TAYLOR, A. L., and M. S. THOMAN, 1964 The genetic map of Escherichia coli K-12. Genetics 90: WALLACE, B. J., and J. PITTARD, 1967 Genetic and biochemical analysis of the isoenzymes concerned in the first reaction of aromatic biosynthesis in Escherichia coli. J. Bacteriol. 93 :