Genetic Basis of Histidine Degradation in Bacillus subtiZis*

SUMMARY Mutants of Bacillus subtilis deficient in urocanase or in imidazolonepropionate hydrolase were isolated. These mutants are unable to use L-histidine as source of carbon or nitrogen for growth. The enzymes are thus essential for the metabolism of histidine. The structural genes specifying these enzymes are closely linked to those specifying the other two enzymes essential for histidine degradation, histidase, and formiminoglutamate hydrolase. The response of these enzymes to induction and catabolite repression is speciCed by a region of the chromosome closely linked to one end of the cluster of structural genes. The gene cluster presumably constitutes an operon. The induction by L-histidine and repression by catabolites of three of the four enzymes required for the degradation of L-histi-dine by Bacillus subtilis have been described earlier papers The the third step in the of degradation, 4-imidazolone-5-propionate H, of hut U, loss of urocanase activity; hut I, loss of IPA hydrolase activity; hut G, loss of FGA hydrolase activity; hut P, pleiotropic loss of the four enzymes of histidine degradation; hut R, insensitivity of the histidine-degrad- ing enzymes to catabolite repression; hut C, constitutive histidine-

These mutants are unable to use L-histidine as source of carbon or nitrogen for growth.
The enzymes are thus essential for the metabolism of histidine. The structural genes specifying these enzymes are closely linked to those specifying the other two enzymes essential for histidine degradation, histidase, and formiminoglutamate hydrolase.
The response of these enzymes to induction and catabolite repression is speciCed by a region of the chromosome closely linked to one end of the cluster of structural genes.
The gene cluster presumably constitutes an operon.
The induction by L-histidine and repression by catabolites of three of the four enzymes required for the degradation of L-histidine by Bacillus subtilis have been described in earlier papers (l-3).
The enzyme catalyzing the third step in the pathway of histidine degradation, 4-imidazolone-5-propionate hydrolase, had not been previously investigated.
The present paper shows that the formation of this enzyme is controlled in the same manner as that of the others. Furthermore, the isolation of mutants lacking IPA* hydrolase and of mutants lacking urocanase permitted the complete mapping of the structural genes and regulatory genes responsible for the controlled degradation of histidine. The results suggest that this cluster of genes constitutes an operon.

EXPERIMENTAL PROCEDURE
Chemicals-In addition to the chemicals described in the preceding paper (3), N-methyl-N-nitrosoguanidine was purchased from Aldrich Chemical Company, and streptomycin sulfate from Squibb.
IPA, a gift of Dr. Peter Scotto, was prepared and purified according to the following procedure (4). Strain N-5 of Aerobacter aerogenes (5), which is deficient in IPA hydrolase, was grown in a medium containing succinic acid as source of carbon and 0.1% imidazolepropionic acid as inducer of of the histidine-degrading enzymes (6). The cells from a 600-ml culture were harvested and sonically disrupted as described previously (7). A 5-ml portion of the extract was added to a solution containing 3.0 g of L-histidine in 100 ml of 0.3 M Tris-HCl buffer, pH 9.0. The mixture was incubated at 37" for 3 hours. At this stage, approximately one-half of the histidine had been converted to urocanic acid and approximately 5% to IPA. The pH was adjusted to 7.4 by the addition of 2 N HCl; the air was evacuated from the reaction flask and replaced by nitrogen.
A lo-ml portion of the cell extract was added to the reaction mixture. The atmosphere of the reaction flask was again repla.ced by nitrogen.
The enzymatic reaction was allowed to proceed at 37" for a period of 90 min. It was arrested by the addition of sufficient 2 N HCl to lower the pH to 1.5. The precipitated protein was removed by centrifugation at 20,000 x g for 30 min. The supernatant was placed on a column (2 x 20 cm) of AG 50-H+ Dowex (200 to 400 mesh) wrapped in aluminum foil in a dark room at 4". The column wa.s then washed with several bed volumes of water and of 0.5 N HCl.
The IPA was eluted with 2 N HCl.
Fractions of 10 ml were collected and examined for ultraviolet extinction at 234 and 260 rnp. The IPA, with a peak extinction at 234 m~.c at a pH of 1 (molar extinction 3500)) was found in tubes 20 through 60. The contents of the tubes were pooled and evaporated to dryness at room temperature in a vacuum.
The residue was dried over PzOs and NaOH and stored at -20".
The yield, estimated spectrophotometrically, based on histidine was 30%.
The material had the characteristic absorption spectrum of IPA (4).
Enzyme Assays-The cultivation of the bacteria has been described (2). To prepare extracts for the assay of the enzymes, the cells from 40 to 50 ml of a culture in the exponential phase of growth were collected by centrifugation, washed with 0.02 M Tris-HCl buffer, pH 8.0, and suspended in 0.95 ml of this buffer. A 0.05-ml portion of a solution containing 4 mg of lysozyme per ml aas added and the suspension was incubated for 10 to 20 min at 37". The mixture was then cooled in an ice bath and the cells were treated at 1.2 to 1.5 amp in an MSE sonicator (Instrumentation Associates) for 1 to 2 min. B O.l-ml portion of a 10% solution of streptomycin sulfate was added to the product of sonic disruption of cells to precipitate nucleic acids. The mixture was kept for 30 min in an ice bath and was then subjected to centrifugation at 22,000 x g at 4" for 20 min. The supernatant was used for the enzyme assays. The concentration of protein in this extract was determined calorimetrically (8). Histidase (I), urocanase (2), and FGA hydrolase (3) were measured by procedures described elsewhere.
IPA hydrolase was assayed spectrophometrically by following

Genetics of Hi&dine Degradation
Vol. 245, No. 14 the rate of disappearance in the absence of oxygen of its extinction at 260 ml* (4,9). A volume of extract containing approximately 1 mg of protein was used. Sufficient 0.1 M potassium phosphate buffer, pH 7.4, to make the sum of the volumes of extract and buffer 2.95 ml was placed in the main compartment of a Thunberg tube fused to a quartz cuvette, and 0.05 ml of 0.015 M IPA in 0.2 N HCl was placed in the top compartment. The tube was evacuated, opened, and the cell extract was added to the main compartment.
The tube was then thoroughly evacuated and incubated at 37" for 10 min. The substrate was mixed with the contents of the main compartment and the decrease in extinction at 260 rnp was recorded in a Gilford recording spectrophotometer at 37". The reaction was linear until approximately 15% of the substrate had disappeared.
Because of the instability of the substrate it was necessary to subtract the decrease in extinction measured in a similar Thunberg tube containing 0.5 to 2 mg of crystalline serum albumin in place of cell extract.
Bacteria-The strains are listed in Table I. The cultivation of the bacteria and the methods used for their transformation have been described (2).
Mutants unable to form urocanase were isolated from cells of the wild strain SH treated with N-methyl-N-nitronitrosoguanidine (10). The cells in which mutagenesis had been induced were plated, at approximately 200 colonies per plate, on agar containing citrate and no nitrogen source other than histidine. Approximately 90 colonies of a total of 5000 showed very poor growth on that medium.
Those failing to grow on plates containing glutamate were eliminated and the remainder were grown One of the strains, SH-7, was chosen for further study.
A mutant defective in IPA hydrolase was obtained by mutagenesis with N-methyl-N-nitronitrosoguanidine (10) of strain SH-11, which forms the other enzymes of histidine degradation constitutively and without sensitivity to catabolite repression (2). In this case the cells in which mutagenesis had been induced were spread on agar plates containing glucose and ammonium sulfate to give 200 colonies per plate. These were replicated to glucose-histidine agar plates. The colonies which failed to reproduce on these plates were tested for histidase and urocanase (11). Of a total of 7000 colonies, 25 failed to grow on the glucosehistidine plates, but possessed both histidase and urocanase. These strains were cultivated in liquid medium containing glucose as major source of carbon and induced with histidine; extracts were prepared and tested for IPA hydrolase and FGA hydrolase.
One was found to be deficient in IPA hydrolase and was designated SH-8. The other mutants lacked FGA hydrolase. Two of them, strains SH-111 and SH-112, were shown to produce material that immunologically cross-reacts with FGA hydrolase (3).

Role and Control of Urocanase and 4-Imidazoloneb-prop&&e
Hydroluse-We examined the levels of the four enzymes of histidine degradation in a variety of mutants.
The results, summarized in Table II, show that the wild strain SH produces the four enzymes when grown in a histidine-containing medium. Strains SH-7 and SH-8, selected for their inability to use L-histidine as a source of nitrogen, are unable to produce, respectively, urocanase and IPA hydrolase. Thus, these two enzymes play essential roles in the conversion of histidine to glutamate and formamide It has been shown previously that in the wild strain SH, histidase, urocanase, and FGA hydrolase are subject to induction by histidine and to repression by catabolites derived from glucose (2). It can be seen that the same controls affect IPA hydrolase. Moreover, mutations to constitutive synthesis and insensitivity to catabolite repression in strain SH-11 affect the control of all four enzymes. Finally, strain SH-5, previously shown to have lost the ability to produce histidase, urocanase, and FGA hydrolase as the result of a single mutation, is also unable to produce IPA hydrolase.
The results presented here and in an earlier paper (2) suggest that the enzymes respond coordinately to induction and repression. Histidine Utilization (hut)-An earlier report has described genetic crosses between mutants deficient in histidase or FGA hydrolase or with altered control of the histidine-degrading enzymes (2). These crosses, carried out by transformation, established the close linkage of these characters. We extended this study using the newly isolated urocanase-or IPA hydrolase-deficient mutants, strains SH-7 and SH-8, as source of transforming DNA. Strain SH-7 was also used as recipient in the transformation experiments; strain SK-8 could not be used in this manner because of its low competence.
A series of crosses between mutants unable to utilize histidine was carried out. Recombinants capable of growth on histidine were selected, as described previously (2). In all of these crosses the recipient was sensitive to erythromycin and the donor resistant. The gene determining response to erythromycin is not linked to the huf genes and was used as reference marker in order to normalize variations in competence of the recipients in the independent experiments (2). This was done by determining in each cross the number of erythromycin-resistant (cry-r) transformants and for each recipient the number of hut+ transformants when DNA from the wild type, strain SH, was used. The results are expressed as the ratio of hut+ over cry-r transformants with mutant DNA divided by the ratio of hut+ over cry-r transformants with wild type DNA.
The results, summarized in Table III, show that hut Hl is more closely linked to hut Ul than to hut II or hut G1; hut Ul is more closely linked to hut II than to hut Gl; hut II is more closely linked to hut Gl than to the other markers.
Finally, hut G& which has been shown to produce material immunologically cross-reacting with FGA hydrolase (3), is very closely linked to hut Gl.
In another series of crosses the linkage of hut Hl, hut Ul, and hut Gl to hut Rl was determined.
The strain carrying the hut Rl mutation can produce the histidine-degrading enzymes in the presence of glucose (2 hut Ul, hut Gl (Table IV).
The experiments presented in Tables III and IV suggest the order hut R, H, U, I, G. We carried out a series of three-factor crosses to determine the order unambiguously. In these crosses, recombinants for two markers were selected and tested for the distribution of the third marker as described previously (2). The  Table V. The reciprocal Crosses 1 and 2 indicate the hut HS is located between hut Rl and hut Ul and the reciprocal Crosses 3 and 4 indicate that hut Ul is located between hut Rl and hut Gl.

Cross 5 places
hut Ul between hut RI and hut II ; it excludes the possibility that hut I1 is located between hut Rl and hut Ul.

Cross 6 indicates
that hut Hl is located between hut Rl and hut I1 and excludes the possibility that hu! II is located between hut Rl and hut HI. Finally, Cross 7 places hut I1 between hut C1, known to be closely linked to hut Rl (2) and hut Gl. It is clear that these results are in complete agreement with the order suggested by the two-factor crosses.