JOURNAL OF BACTERIOLOGY, Jan. 1975, p. 29-35 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 1
Printed in U.S.A.
Regulation of Arylsulfatase Synthesis by Sulfur Compounds in Klebsiella aerogenes TOSHIRO ADACHI, YOSHIKATSU MUROOKA, AND TUKUYA HARADA* The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, Japan Received for publication 19 August 1974
In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys-), and mutants (AtsS-, AtsR-) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS- mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS- mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR- mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.
Harada and Spencer (8) showed that, in many fungi, arylsulfatase synthesis is inhibited by the presence of sulfur compounds, such as sulfate, sulfite, thiosulfate, and cysteine, which are thought to be direct intermediates in the assimilation of sulfate, but that it is not inhibited by compounds such as methionine, taurine, and cysteate, which are not direct intermediates. A similar division of sulfur sources was observed in the arylsulfatase synthesis of Aerobacter aerogenes (9). The effects of various sulfur compounds in repressing arylsulfatase synthesis has been thought to be due to conversion of these compounds to a single corepressor compound: inorganic sulfate in the case of fungi and cysteine in the case of A. aerogenes. The work described in this paper indicates that there may be two independent functional corepressors of arylsulfatase synthesis in Klebsiella aerogenes W70.
Isolation of mutants. N-methyl-N'-nitro-Nnitrosoguanidine was used as a mutagenic agent, as described by Adelberg et al. (3). The mutagenized cells were treated with penicillin G (3,000 units/ml) for enrichment of auxotrophs. For isolation of mutants in which control of arylsulfatase synthesis was altered, cells were spread on agar plates of selective minimal medium containing 1 mM sodium sulfate or cysteine as the sole source of sulfur. Two screening techniques were used. One was the test paper method described by Harada (7) using nitrocatechol sulfate as a chromogenic substrate for arylsulfatase. In brief, filter paper is dipped into a 0.01 M solution of nitrocatechol sulfate and then dried at room temperature. The test paper is placed on colonies grown on agar medium. The hydrolase activity of the test organism can be determined by measuring the time required for development of a color due to hydrolysis. The other technique depended on the fact that colonies with arylsulfatase activity stained blue in the presence of indoxylsulfate, a substrate for arylsulfatase, whereas colonies lacking the enzyme remained colorless. For the latter method the screening plates contained 1 mM potassium indoxylsulfate and 0.025% lignin which reacts with indoxyl forming a red pigment under anaerobic and neutral or weakly alkaline conditions (10) and increases the blue staining of colonies under neutral conditions. Repression technique. An overnight culture in minimal medium containing methionine as the sole source of sulfur was diluted with fresh medium of the same composition to give a cell density of approxi-
MATERIALS AND METHODS Bacterial strains and growth conditions. The strains employed in this work are listed in Table 1. They are derivatives of K. aerogenes W70 described by MacPhee et al. (11). Organisms were cultured as described previously (1), except that 50 1sg of L-leucine, L-cysteine, or both were added per mg when required, and cells were grown at 37 C. 29
ADACHI, MUROOKA, AND HARADA
30
TABLE 1. Bacterial strains Strain
Phenotypea
Derivation
W70
Wild type
See MacPhee et al.
K13 K17 K103 K152 K170 K501 K172 K31-1
Mao-
See Adachi et al. (1) Mutagenesis of W70 Mutagenesis of K13 Mutagenesis of K103 Mutagenesis of K17 Mutagenesis of K13 Mutagenesis of K501 Mutagenesis of K17
(1 1) TyrNMao-, LeuMao , Leu-, Cys TyrN-, CysAMao-, AtsSMao-, AtsS-, CysTyrN-, AtsR-
a Mao-, Monoamine oxidase negative; AtsS-, synthesis of arylsulfatase in the presence of inorganic sulfate and repressed synthesis in the presence of cysteine; AtsR-, constitutive synthesis of arylsulfatase in the presence of inorganic sulfate or cysteine; Cys-, requirement for cysteine; CysA-, sulfate-thiosulfate permease negative; TyrN-, inability to grow on tyramine as a nitrogen.
mately 1.5 Klett units (approximately 2 x 107 cells per ml). The culture was incubated at 37 C and, when the cell concentration reached about 15 Klett units, the culture was divided into several portions and one sulfur compound was added to each. The resulting cultures were aerated at 37 C, and samples were taken at intervals. The arylsulfatase levels in long-term experiments were measured as described previously
(1).
Determination of arylsulfatase activity. Arylsulfatase activity was assayed in whole cells as reported previously (1). Cells were collected on a Whatman glass fiber filter GF/B and washed with 0.1 M
tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2). The filters were dipped in the same buffer (1 ml) and preincubated for 5 min at 30 C before adding warm p-nitrophenlysulfate in the same buffer (3 ml) at a final concentration of 2.5 mM. The mixtures were incubated at 30 C for 5 to 60 min, depending on the activity present, and then the reaction was terminated by adding 1 ml of 1 N NaOH containing 0.1 M Na,PO4, since phosphate ion is a strong inhibitor of arylsulfatase (11). The filters and cells were removed by centrifugation at 3,000 rpm for 20 min, and the optical density of the yellow color developed was measured in a spectrophotometer at 400 nm. The amount of p-nitrophenol liberated was calculated from a calibration curve. As a control, cells on glass fibers and substrate were incubated separately and combined immediately before addition of NaOH and Na,PO4. The cell concentration was determined by measuring turbidity with a KlettSummerson colorimeter, using a no. 66 filter, which was calibrated by dry weight determination. One unit of activity was taken as the amount causing formation of 1 gmol of p-nitrophenol per min, at 30 C. Inorganic sulfate and cysteine transport. Cells growing exponentially in minimal medium containing methionine as the sole source of sulfur were harvested, washed twice with sulfur-free minimal medium at
J. BACTERIOL.
5 C, and resuspended in sulfur-free minimal medium containing 50 gg of chloramphenicol per ml. To establish the initial rate of uptake, a sample of cell suspension (0.7 mg dry weight) was rapidly pipetted into a prewarmed minimal medium containing 10 to 100 gM inorganic sulfate, 50 jig of chloramphenicol per ml, and 2 x 105 counts/min or [3SJSSO,-2 (final volume, 0.5 ml). The initial rates of uptake were calculated from the linear uptakes determined at 30-s intervals for 2 min at 30 C. The incubation was terminated by filtering the cell suspension through a membrane filter (0.45 jm pore size, Toyo Roshi Co.). Cells on the membrane filter were washed quickly with 5 ml of cold sugar- and sulfur-free minimal
medium. Inorganic sulfate transported into the cells was rapidly lost on rinsing the cells with sugar- or sulfur-containing minimal medium. Cellular radioactivity was measured in a Beckman scintillation spectrometer, LS-100. For assay of cysteine transport, cells were grown in minimal medium containing cysteine as the sole source of sulfur. As substrate of transport, cysteine was used in place of inorganic sulfate. The radioactivity in the assay mixture was 1.5 x 106 counts/min. Measurement of cysteine formation. Cells growing exponentially in minimal medium containing methionine as the sole source of sulfur were harvested and washed twice with sulfur-free minimal medium. Cell samples (2 to 3 mg dry weight) were incubated for 30 min at 37 C in 0.1 ml (final volume) of minimal medium containing 100 uM Na2SO4 and 7 x 106 counts/min of [35S]sulfate with or without 100 AM methionine. The methods for extraction of intracellular cysteine and for two-dimensional paper chromatography of the N-ethylmaleimide (NEM) derivative of cysteine were used as described by Ellis (6). The paper chromatogram was exposed to X-ray film for 3 weeks. The area corresponding to the NEM-cysteine adduct was cut out and its radioactivity was determined in a Beckman scintillation spectrometer, LS-100. Chemicals. [35S ]Na2SO4 and [35S ]cysteine were obtained from the Radiochemical Centre, Amersham. p-Nitrophenylsulfate was obtained from Sigma Chemical Co. and recrystallized from alkaline aqueous ethanol. Other chemicals were of the purest grade available commercially.
RESULTS Some characteristics of cysteine auxotrophs. Inorganic sulfate is thought to be metabolized to cysteine via sulfite, and sulfide or thiosulfate in microorganisms (5, 13, 15). To restrict the identity of functional corepressor of arylsulfatase synthesis in K. aerogenes, many cysteine auxotrophs were isolated from a monoamine oxidase-negative (Mao-) mutant (2) or TyrN - mutant which could not use tryamine as the sole source of nitrogen. Mutants were tested for their ability to grow with sulfate, sulfite, sulfide, thiosulfate, cysteine, or methionine as the sole source of sulfur, and for their ability to
VOL. 121, 1975
31
REGULATION OF ARYLSULFATASE BY SULFUR
take up inorganic sulfate into the cells (Table 2). To test repression of enzyme synthesis, two types of mutant strains were selected: one (strain K170) which could not transport inorganic sulfate and the other (strain K152) which could utilize sulfide and thiosulfate but not sulfite as the sole source of sulfur. K172 shown in Table 2 was derived from mutant K501 m 0~~~~~~~ (described later) in which regulation of arylsulfatase synthesis was altered. Repression of arylsulfatase synthesis in E j cysteine auxotrophs by sulfur compounds. Inorganic sulfate may be a corepressor of aryl- U grow with inorganic sulfate or sulfite as the sole source of sulfur, was completely repressed when inorganic sulfate, sulfite, sulfide, thiosulfate, or cysteine was added during growth with methio20 30 10 0 nine (Fig. 1). Results obtained in a long-term CULTURE DENSITY (KLETT UNITS) experiment (Table 3), after four doublings of FIG. 1. Kinetics of arylsulfatase repression in cells growing on each sulfur compound as the strain K152 by various sulfur compounds. Cells were sulfur source, were consistent with short-term incubated in a minimal medium containing methiokinetic data. Strain K152 chould not synthesize nine (1 mM) as the sole source of sulfur. Sulfur cysteine from inorganic sulfate, since it is auxo- compounds (1 mM) were added at the time indicated trophic for cysteine. Thus, repression of arylsul- by an arrow. Addition: 0, none; A, NaSO4; fatase synthesis in this mutant by inorganic Na.SO.; Na2S; A, Na2S20,; 0, cysteine. sulfate was not due to conversion of inorganic sulfate to cysteine. It was demonstrated that K170 which could not grow with sulfate as the the ability of strain K152 to synthesize cysteine sulfur source (Table 2), synthesized arylsulfafrom inorganic sulfate was in fact far less than tase in the presence of inorganic sulfate, sulfite, that of strain K17 (Table 4). In both strains, or thiosulfate but not in the presence of cysteine cysteine accumulation was slightly stimulated (Table 3). Cells of this mutant strain could not by addition of methionine. Thus, the repression take up sulfate (Table 2). Although the characof arylsulfatase synthesis by inorganic sulfate teristics of the transport system for inorganic was not due to its conversion to cysteine, sulfate in K. aerogenes have not yet been sulfide, or thiosulfate. On the other hand, it described, in Salmonella typhimurium (14) the seemed likely that exogenous cysteine could sulfate transport system transports sulfite and repress enzyme synthesis in either of two ways: thiosulfate in addition to inorganic sulfate. In (i) it might cause repression itself or after K. aerogenes, as in S. typhimurium (4), the conversion to a corepressor related to cysteine, transport system was strongly inhibited by or (ii) it might cause repression after degradasulfite or thiosulfate, whereas other sulfur comtion to inorganic sulfate. pounds, such as sulfide, cysteine, and methioAnother type of cysteine auxotroph, strain nine stimulated inorganic sulfate transport TABLE 2. Growth responses of cysteine auxotrophs to various sulfur compounds,a and inorganic sulfate transporting activities of the mutants Wi
0,
U,
Growth (mg [dry wt I cell/ml) Strain
_
Na,SO4 K17 K152 K170 K172 a
Inorganic sulfate
transport___
4.82
Vol. 121, No. 1
Printed in U.S.A.
Regulation of Arylsulfatase Synthesis by Sulfur Compounds in Klebsiella aerogenes TOSHIRO ADACHI, YOSHIKATSU MUROOKA, AND TUKUYA HARADA* The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, Japan Received for publication 19 August 1974
In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys-), and mutants (AtsS-, AtsR-) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS- mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS- mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR- mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.
Harada and Spencer (8) showed that, in many fungi, arylsulfatase synthesis is inhibited by the presence of sulfur compounds, such as sulfate, sulfite, thiosulfate, and cysteine, which are thought to be direct intermediates in the assimilation of sulfate, but that it is not inhibited by compounds such as methionine, taurine, and cysteate, which are not direct intermediates. A similar division of sulfur sources was observed in the arylsulfatase synthesis of Aerobacter aerogenes (9). The effects of various sulfur compounds in repressing arylsulfatase synthesis has been thought to be due to conversion of these compounds to a single corepressor compound: inorganic sulfate in the case of fungi and cysteine in the case of A. aerogenes. The work described in this paper indicates that there may be two independent functional corepressors of arylsulfatase synthesis in Klebsiella aerogenes W70.
Isolation of mutants. N-methyl-N'-nitro-Nnitrosoguanidine was used as a mutagenic agent, as described by Adelberg et al. (3). The mutagenized cells were treated with penicillin G (3,000 units/ml) for enrichment of auxotrophs. For isolation of mutants in which control of arylsulfatase synthesis was altered, cells were spread on agar plates of selective minimal medium containing 1 mM sodium sulfate or cysteine as the sole source of sulfur. Two screening techniques were used. One was the test paper method described by Harada (7) using nitrocatechol sulfate as a chromogenic substrate for arylsulfatase. In brief, filter paper is dipped into a 0.01 M solution of nitrocatechol sulfate and then dried at room temperature. The test paper is placed on colonies grown on agar medium. The hydrolase activity of the test organism can be determined by measuring the time required for development of a color due to hydrolysis. The other technique depended on the fact that colonies with arylsulfatase activity stained blue in the presence of indoxylsulfate, a substrate for arylsulfatase, whereas colonies lacking the enzyme remained colorless. For the latter method the screening plates contained 1 mM potassium indoxylsulfate and 0.025% lignin which reacts with indoxyl forming a red pigment under anaerobic and neutral or weakly alkaline conditions (10) and increases the blue staining of colonies under neutral conditions. Repression technique. An overnight culture in minimal medium containing methionine as the sole source of sulfur was diluted with fresh medium of the same composition to give a cell density of approxi-
MATERIALS AND METHODS Bacterial strains and growth conditions. The strains employed in this work are listed in Table 1. They are derivatives of K. aerogenes W70 described by MacPhee et al. (11). Organisms were cultured as described previously (1), except that 50 1sg of L-leucine, L-cysteine, or both were added per mg when required, and cells were grown at 37 C. 29
ADACHI, MUROOKA, AND HARADA
30
TABLE 1. Bacterial strains Strain
Phenotypea
Derivation
W70
Wild type
See MacPhee et al.
K13 K17 K103 K152 K170 K501 K172 K31-1
Mao-
See Adachi et al. (1) Mutagenesis of W70 Mutagenesis of K13 Mutagenesis of K103 Mutagenesis of K17 Mutagenesis of K13 Mutagenesis of K501 Mutagenesis of K17
(1 1) TyrNMao-, LeuMao , Leu-, Cys TyrN-, CysAMao-, AtsSMao-, AtsS-, CysTyrN-, AtsR-
a Mao-, Monoamine oxidase negative; AtsS-, synthesis of arylsulfatase in the presence of inorganic sulfate and repressed synthesis in the presence of cysteine; AtsR-, constitutive synthesis of arylsulfatase in the presence of inorganic sulfate or cysteine; Cys-, requirement for cysteine; CysA-, sulfate-thiosulfate permease negative; TyrN-, inability to grow on tyramine as a nitrogen.
mately 1.5 Klett units (approximately 2 x 107 cells per ml). The culture was incubated at 37 C and, when the cell concentration reached about 15 Klett units, the culture was divided into several portions and one sulfur compound was added to each. The resulting cultures were aerated at 37 C, and samples were taken at intervals. The arylsulfatase levels in long-term experiments were measured as described previously
(1).
Determination of arylsulfatase activity. Arylsulfatase activity was assayed in whole cells as reported previously (1). Cells were collected on a Whatman glass fiber filter GF/B and washed with 0.1 M
tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2). The filters were dipped in the same buffer (1 ml) and preincubated for 5 min at 30 C before adding warm p-nitrophenlysulfate in the same buffer (3 ml) at a final concentration of 2.5 mM. The mixtures were incubated at 30 C for 5 to 60 min, depending on the activity present, and then the reaction was terminated by adding 1 ml of 1 N NaOH containing 0.1 M Na,PO4, since phosphate ion is a strong inhibitor of arylsulfatase (11). The filters and cells were removed by centrifugation at 3,000 rpm for 20 min, and the optical density of the yellow color developed was measured in a spectrophotometer at 400 nm. The amount of p-nitrophenol liberated was calculated from a calibration curve. As a control, cells on glass fibers and substrate were incubated separately and combined immediately before addition of NaOH and Na,PO4. The cell concentration was determined by measuring turbidity with a KlettSummerson colorimeter, using a no. 66 filter, which was calibrated by dry weight determination. One unit of activity was taken as the amount causing formation of 1 gmol of p-nitrophenol per min, at 30 C. Inorganic sulfate and cysteine transport. Cells growing exponentially in minimal medium containing methionine as the sole source of sulfur were harvested, washed twice with sulfur-free minimal medium at
J. BACTERIOL.
5 C, and resuspended in sulfur-free minimal medium containing 50 gg of chloramphenicol per ml. To establish the initial rate of uptake, a sample of cell suspension (0.7 mg dry weight) was rapidly pipetted into a prewarmed minimal medium containing 10 to 100 gM inorganic sulfate, 50 jig of chloramphenicol per ml, and 2 x 105 counts/min or [3SJSSO,-2 (final volume, 0.5 ml). The initial rates of uptake were calculated from the linear uptakes determined at 30-s intervals for 2 min at 30 C. The incubation was terminated by filtering the cell suspension through a membrane filter (0.45 jm pore size, Toyo Roshi Co.). Cells on the membrane filter were washed quickly with 5 ml of cold sugar- and sulfur-free minimal
medium. Inorganic sulfate transported into the cells was rapidly lost on rinsing the cells with sugar- or sulfur-containing minimal medium. Cellular radioactivity was measured in a Beckman scintillation spectrometer, LS-100. For assay of cysteine transport, cells were grown in minimal medium containing cysteine as the sole source of sulfur. As substrate of transport, cysteine was used in place of inorganic sulfate. The radioactivity in the assay mixture was 1.5 x 106 counts/min. Measurement of cysteine formation. Cells growing exponentially in minimal medium containing methionine as the sole source of sulfur were harvested and washed twice with sulfur-free minimal medium. Cell samples (2 to 3 mg dry weight) were incubated for 30 min at 37 C in 0.1 ml (final volume) of minimal medium containing 100 uM Na2SO4 and 7 x 106 counts/min of [35S]sulfate with or without 100 AM methionine. The methods for extraction of intracellular cysteine and for two-dimensional paper chromatography of the N-ethylmaleimide (NEM) derivative of cysteine were used as described by Ellis (6). The paper chromatogram was exposed to X-ray film for 3 weeks. The area corresponding to the NEM-cysteine adduct was cut out and its radioactivity was determined in a Beckman scintillation spectrometer, LS-100. Chemicals. [35S ]Na2SO4 and [35S ]cysteine were obtained from the Radiochemical Centre, Amersham. p-Nitrophenylsulfate was obtained from Sigma Chemical Co. and recrystallized from alkaline aqueous ethanol. Other chemicals were of the purest grade available commercially.
RESULTS Some characteristics of cysteine auxotrophs. Inorganic sulfate is thought to be metabolized to cysteine via sulfite, and sulfide or thiosulfate in microorganisms (5, 13, 15). To restrict the identity of functional corepressor of arylsulfatase synthesis in K. aerogenes, many cysteine auxotrophs were isolated from a monoamine oxidase-negative (Mao-) mutant (2) or TyrN - mutant which could not use tryamine as the sole source of nitrogen. Mutants were tested for their ability to grow with sulfate, sulfite, sulfide, thiosulfate, cysteine, or methionine as the sole source of sulfur, and for their ability to
VOL. 121, 1975
31
REGULATION OF ARYLSULFATASE BY SULFUR
take up inorganic sulfate into the cells (Table 2). To test repression of enzyme synthesis, two types of mutant strains were selected: one (strain K170) which could not transport inorganic sulfate and the other (strain K152) which could utilize sulfide and thiosulfate but not sulfite as the sole source of sulfur. K172 shown in Table 2 was derived from mutant K501 m 0~~~~~~~ (described later) in which regulation of arylsulfatase synthesis was altered. Repression of arylsulfatase synthesis in E j cysteine auxotrophs by sulfur compounds. Inorganic sulfate may be a corepressor of aryl- U grow with inorganic sulfate or sulfite as the sole source of sulfur, was completely repressed when inorganic sulfate, sulfite, sulfide, thiosulfate, or cysteine was added during growth with methio20 30 10 0 nine (Fig. 1). Results obtained in a long-term CULTURE DENSITY (KLETT UNITS) experiment (Table 3), after four doublings of FIG. 1. Kinetics of arylsulfatase repression in cells growing on each sulfur compound as the strain K152 by various sulfur compounds. Cells were sulfur source, were consistent with short-term incubated in a minimal medium containing methiokinetic data. Strain K152 chould not synthesize nine (1 mM) as the sole source of sulfur. Sulfur cysteine from inorganic sulfate, since it is auxo- compounds (1 mM) were added at the time indicated trophic for cysteine. Thus, repression of arylsul- by an arrow. Addition: 0, none; A, NaSO4; fatase synthesis in this mutant by inorganic Na.SO.; Na2S; A, Na2S20,; 0, cysteine. sulfate was not due to conversion of inorganic sulfate to cysteine. It was demonstrated that K170 which could not grow with sulfate as the the ability of strain K152 to synthesize cysteine sulfur source (Table 2), synthesized arylsulfafrom inorganic sulfate was in fact far less than tase in the presence of inorganic sulfate, sulfite, that of strain K17 (Table 4). In both strains, or thiosulfate but not in the presence of cysteine cysteine accumulation was slightly stimulated (Table 3). Cells of this mutant strain could not by addition of methionine. Thus, the repression take up sulfate (Table 2). Although the characof arylsulfatase synthesis by inorganic sulfate teristics of the transport system for inorganic was not due to its conversion to cysteine, sulfate in K. aerogenes have not yet been sulfide, or thiosulfate. On the other hand, it described, in Salmonella typhimurium (14) the seemed likely that exogenous cysteine could sulfate transport system transports sulfite and repress enzyme synthesis in either of two ways: thiosulfate in addition to inorganic sulfate. In (i) it might cause repression itself or after K. aerogenes, as in S. typhimurium (4), the conversion to a corepressor related to cysteine, transport system was strongly inhibited by or (ii) it might cause repression after degradasulfite or thiosulfate, whereas other sulfur comtion to inorganic sulfate. pounds, such as sulfide, cysteine, and methioAnother type of cysteine auxotroph, strain nine stimulated inorganic sulfate transport TABLE 2. Growth responses of cysteine auxotrophs to various sulfur compounds,a and inorganic sulfate transporting activities of the mutants Wi
0,
U,
Growth (mg [dry wt I cell/ml) Strain
_
Na,SO4 K17 K152 K170 K172 a
Inorganic sulfate
transport___
4.82
