169 , No. 6 The phs G ene and Hydrogen Sulfide Production Salmonella typhimurium by MARTA A. CLARK AND ERICKA L. BARRETT * Department of Food Science and Technology , University of California , Davis , California 95616 Received 1 December 1986/Accepted 5 March 1987 Salmonella typhimurium produces H2S from thiosulfate or sulfite .
The respective pathways for the two reductions must be distinct as mutants carrying motations in phs , chi , and menB reduced sulfite , but not thiosulfate , to H2S , and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite .
The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobicafly grown wild-type cultures .
A number of hydroxylamine , transposon TnlO insertion , and Mu dl ( Apr lac ) operon fusion mutants defective in phs were characterized .
One of the hydroxylamine mutants was an amber mutant , as indicated by suppression of its mutation in a supD background .
The temperature-sensitive phs mutants produced 112S and methyl viologen-linked thiosulfate reductase at 30 °C but not at 42 °C .
The reductases in all such mutants grown at 300C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility , suggesting that phs is not the structural gene for thiosulfate reductase .
Expression of P-galactosidase in phs : : Mu dl ( Apr lac ) mutants was dependent on anaerobiosis and the presence of reduced sulfur .
It was also strongly influenced by carbon source and growth stage .
The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide .
Although the production of hydrogen sulfide from thiosulfate is a characteristic of prime importance in the identification of Salmonella spp. , little is known about the biochemistry and genetics of this system .
A gene essential for this activity , phs , has been mapped ( 35 , 36 ) , but the relationship between phs expression and H2S production has not been examined .
By analogy with the sulfate-reducing bacteria , one might surmise that thiosulfate is an electron-acceptor for anaerobic respiration by Salmonella spp. .
In fact , the process of H2S production from thiosulfate does share many features with other anaerobic respiratory systems in Salmonella spp. .
For example , the process is lacking in pleiotropic chlorate-resistant mutants which exhibit many defects in anaerobic energy metabolism ( 16 , 30 ) , and it is negatively affected by air ( 1 ) .
Furthermore , like trimethylamine oxide reduction , H2S production is defective in menaquinone mutants ( 16 , 18 ) and in wide-type Salmonella typhimurium grown anaerobically with nitrate ( D. L. Riggs and E. L. Barrett , Abstr .
On the other hand , H2S production from thiosulfate by other members of the family Enterobacteriaceae , namely Proteus mirabilis and Citrobacterfreundii , has been suggested to be a relatively unimportant secondary activity of the anaerobic respiratory enzyme that reduces tetrathionate to thiosulfate ( 15 , 25 , 26 ) .
S. typhimurium is also capable of tetrathionate reduction ( 7 , 26 ) , but even less is known about this anaerobic activity in Salmonella spp. .
except that tetrathionate reductase is also a molybdoenzyme ( 12 ) .
produce H2S from sulfite as well as from thiosulfate and are thus capable of reducing both sulfur atoms in thiosulfate to sulfide ( 23 ) .
However , the fact that among the Enterobacteriaceae , only Salmonella and Edwardsiella positive for H2S production media spp. .
are on containing sulfite , whereas positive many genera are on thiosulfate-containing media ( 27 ) , suggests that the two pathways for H2S production may be distinct .
We have initiated a study of H2S production by S. typhimurium .
The results presented here indicate that the pathways for thiosulfate and sulfite reduction are , in fact , distinct and that thiosulfate reduction differs substantially from the other well-characterized anaerobic reductions in S. typhimurium and Escherichia coli .
MATERIALS AND METHODS Strains , media , and growth-conditions .
The strains of S. typhimurium used in these studies are listed in Table 1 .
Nutrient agar , nutrient broth , peptone iron ( PI ) agar , and tryptone were all from Difco Laboratories .
When used in plates , PI agar always included an overlay of the same medium .
Unless otherwise indicated , H2S production was detected in nutrient agar containing 1 mM FeCl2 and either 6 mM Na2S203 or 12 mM Na2SO3 .
The mineral base used in most minimal media was Vogel and Bonner medium E ( VBC ) ( 34 ) formulated with 50 mM phosphate buffer ( pH 6.6 ) and supplemented with 1.0 mM CaCl2 , 1.0 pM each ZnCl2 , FeSO4 , Na2Se203 , and Na2MoO4 , and 0.1 p , M each MnSO4 , CuS04 , and Co ( NO3 ) 2 .
Unless otherwise noted , the medium contained 5.6 mM glucose or galactose as the carbon source and 3mN Na2S203 .
In some experiments , M9 mineral base ( 9 ) was used instead of VBC .
Green plates and Luria-Bertani ( LB ) broth were described by Miller ( 24 ) .
All sugars used were D-isomers .
Anaerobic-growth-conditions for carbohydrate growth-rate studies and for thiosulfate reductase assays were achieved by filling tubes or flasks to the top and stoppering them after inoculation .
Aerobic conditions were achieved by incubating partially filled tubes on a roller drum .
All incubations were done at 37 °C unless otherwise noted .
All Phs-mutants were isolated as white colonies on PI agar .
The phs : : TnlO insertion mutants were 60 of ml on isolated PI agar containing p , g tetracycline per with a random pool of TnlO insertions ( 9 ) kindly provided by John Roth .
Mu dl ( Apr lac ) fusion mutants were generated with a P22 lysate of strain L1119 as described previously ( 16 ) 239 P22 ( NK186 ) x LT2 This study SGSC P22 ( TN2063 ) EB222 x This study EB244 phs-118 ( Am ) EB245 cysB403 EB246 phs-1O1 : : Mu dl ( Apr lac ) oxrA2 : : TnlO EB299 H2S - ( genotype not yet determined ) a Indicates , e.g. , P22 lysate grown on strain TN2063 use ( d to infect strain LT2 .
b Salmonella Genetic Stock Centre , care of K. E. Sander , on PI agar containing 50 p.g of ampicillin per ml .
Mutants were purified on green plates , and Mu dl inserrtion mutants were immobilized as described previously ( 4 [ ) .
When the insertion mutants were corrected by using transiducing phage grown on strain TT513 , Phs + was 100 % liinked to the inheritance of Lac-and Amps .
Localized mut ; agenesis ( 13 ) was used in three different ways to obtain point phs mutants : ( i ) P22 grown on LT2 was mutagenized and used to infect EB217 ( hisG46 ) , and His + transductants were screened for lack of H2S production ( H2S5 ) on PI agar ; ( ii ) IP22 grown on LT2 was mutagenized and used to infect TT513 ( zec-2 : : TnlO , insertion 80 % linked to phs in CP22 ) on modified Bochner agar ( 22 ) , and Tet5 transductants were screened for H2S on PI agar ; and ( iii ) P22 grown on TT513 vvas mutagen-ized and used to infect LT2 on nutrient agatr containing tetracycline ( 20 , ug/ml ) , and Tetr H2S colon : y types were isolated .
Transductions were performed by ths e methods of Ely et al. ( 11 ) with P22 int4 ( 29 ) .
Cotransduction of phs : : TnJO with his was determined by selecting histidine prototrophs from EB217 infected with phage P22 grown on the phs : : TnJO mutant and scoring for coinheritance of tetracycline resistance .
Cotransduction of phs : : Mu dl insertions with his was determined by selecting tetracycline-resistant transductants from the phs : : Mu dl infected with P22 grown on EB235 and scoring for coinherit-ance of the ability to produce H2S ( H2S + ) .
H2S production was tested by inoculating 5 ml of melted detection medium in a tube ( 13 by 100 mm ) with 0.1 ml of culture grown overnight in LB broth and observing for blackening within 24 h. For the thiosulfate reductase assay , thiosulfate-containing minimal media were inoculated at a 2 % concentration with cells grown overnight in the same medium and incubated anaerobically to the early stationary-phase .
Harvested cells were washed twice in 50 mM phosphate buffer ( pH 7.2 ) and frozen at -80 °C .
The pellets were suspended in the same buffer containing 5 mM MgCl2 , 0.1 M sucrose , and 1 mM phenylmethylsulfonyl fluoride , transferred to 10-ml polyeth-ylene centrifuge tubes fitted with serum caps , and gassed with N2 .
Cells were broken by two rounds of freezing in a dry ice-ethanol bath , followed by thawing under hot ( 60 °C ) running water .
The broken cells were assayed by a modification of the methyl viologen-linked thiosulfate reductase assay of Badziong and Thauer designed for Desulfovibrio vulgaris ( 2 ) .
The assay was performed in a Varian DMS 100 UV-visible spectrophotometer with a slit width of 1.0 nm ; E578 for methyl viologen was 10,900 cm-1 M-1 .
Serum cap cuvettes were completely filled with 5 mM methyl viologen in 100 mM sodium phosphate buffer ( pH 7.4 ) , and oxygen was scavenged by adding 13 j ± l of fresh dithionite solution ( 0.25 M Na2S204 in 0.52 M NaHCO3 ) .
Cell extract was added , and the methyl viologen was reduced to an A578 of 1.8 to 2.0 with additional dithionite solution .
After measurement of the rate of endogenous methyl viologen oxidation , the reaction was started by the addition of 10 , ul of 1.9 M Na2S203 , and the decrease in A578 was followed for several minutes .
One unit of specific activity represents the oxidation of 2 , umol of methyl viologen per min per mg of protein .
Each reported specific activity is the of three or average more determinations .
Standard deviations in all cases were less than 10 % .
Protein was determined by the Bradford method ( 6 ) .
For the P-galactosidase assays , cultures were all harvested at an OD650 of 0.1 .
P-Galactosidase was assayed by the method of Miller , in which specific activity is defined as nanomoles of o-nitrophenol produced per minute per OD600 units at 28 °C ( 24 ) .
Each reported specific activity is the average of three determinations .
Standard deviations in all cases were less than 5 % .
For electrophoresis , broken cells were centrifuged at 27,000 x g for 20 min , suspended to a protein concentration of 2 to 4 mg/ml in 0.1 M Tris buffer ( pH 7.2 ) containing 10 % ( wt/vol ) sucrose , 2 % Triton X-100 , and 1 mM phenylmethylsulfonyl fluoride , gassed with N2 , and then stirred for 45 min on ice .
The suspension was centrifuged as before , and the supernatant was concentrated in Spectrapor ( 12,000 to 14,000 molecular weight cutoff ) dialysis tubing surrounded by polyethylene-glycol .
Slab gels used to locate thiosulfate reductase consisted of a 6 % polyacrylamide separating gel containing 0.375 M Tris ( pH 8.8 ) and 0.1 % Triton X-100 , and a stacking gel of 3 % polyacrylamide with 0.125 M Tris ( pH 6.8 ) and 0.1 % Triton X-100 .
Bromphenol blue was used as the tracking dye .
Thiosulfate reductase was stained by the method of Lun Source LT2 B. N. Ames G. Wilcox L1119 M. J. Voll N. Kleckner A. SAsirman C. G. Miller CP22 NK186 SAST66 TN2063 J. R. Roth J. R. Roth J. R. Roth G. W. Chang TT34 TT513 TT7610 TC99 G. W. Chang TC11O J. R. Roth P22 ( TN2063 ) x LT2a SGSCb TShTiS s Cstudy This study EB217 hisG46 EB218 cysI68 EB222 phs-1O1 : : Mu dl ( Apr lac ) EB223 phs-102 ( Ts ) EB224 through zec-2 : : TnlO phs-103 ( Ts ) through EB229 zec-2 : : TnlO phs-108 ( Ts ) EB230 cysJ299 araB9 EB231 phs-109 : : TnlO EB232 cysG458 EB233 phs-11O : : Mu dl ( Apr lac ) EB234 phs-l11 : : Mu dl ( Apr lac ) EB235 hisD : : TnlO SGSC This study SGSC This study This study LT2 This study EB236 through phs-112 : : Mu dl ( Apr lac ) EB241 through phs-117 : : Mu dl ( Apr lac ) EB242 cysA : : TnlO P22 ( NK186 ) x LT2 This study SGSC P22 ( TN2063 ) EB222 x This study EB244 phs-118 ( Am ) EB245 cysB403 L. 161 ~ ~ Y M R H 9 , 1987 O69L98. ,7 ~ PHH ~ I ~ U ~ I ~ UM S2S.PRODUCTION 2393 VOV T TABLE 2 .
Production of H2S from thiosulfate and suffite Strain ( relevant Supplement ( 10 mM ) to H2prdcinfom genotype ) nutrient agar Thiosulfate Sulfite LT2 ( wild type ) None + + - Glucose - + + Galactose + + + Glycerol + + - KNO -- KNO3 and galactose Trimethylamine oxide + Trimethylamine oxide + + and galactose None - + + Galactose - + + a H25 production was tested in nutrient agar containing 1 mM FeCI2 and either 6 mM Na2S2O3 12 mM Na2S03 , A positive consisted of or test blackening due to the accumulatiQn of ferrous-sulfide .
No H25 production was observed in the absence of added thiosulfate , or sulfite .
and DeMoss ( 21 ) designed for nitrate reductase , except that 0.15 M Na2S2O3 ( final concentration ) was used in place of nitrate .
3 ¬ + + CP22 ( phs ) RESULTS H2S production from thiosulfate and sulfite .
Wild-type S. typhimurium and a phs mutant isolated previously ( 36 ) were tested for the ability to produce H2S from thiosulfate and sulfite ( Table 2 ) .
Thiosulfate and sulfite reduction were favored under different conditions , and phs was essential only for the reduction of thiosulfate .
Sulfite reduction required a fermentable carbon source , while thiosulfate reduction , in contrast , appeared to be rep-ressed by glucos ` e .
The two reductions did show a common response to the presence of other electron-acceptors .
The addition of 10 mM nitrate prevented H2S production from both sulfur compounds , while trimethylamine oxide had no repressive effect .
Results in minimal-medium ( not shown ) paralleled those in nutrient agar .
Cysteine did not interfere with H2S production , indicating that H2S production is a dissimnilatory pathway regulated independently of the biosynthetic need for reduced sulfur .
Additional differences between the respective enzyme systems for thiosulfate and sulfite reduction were revealed in tests of other types of mutants .
A menB mutant ( strain TC99 ) produced H2S from sulfite but not from thiosulfate , while a ubi mutant ( strain SAST66 ) produced H25 from both compounds .
A chiA ( TC110 ) mutant was negative for H25 from thiosulfate , in agreement with previous reports ( 16 , 30 ) , but it did produce H2S froM Sulfite , suggesting that thiosulfate reduction , but not suffite reduction , involves a molybdo-enzyme .
The involvement of heme in both activities was indicated by the absence of H2S production fromn either substrate in a hemA mutant ( TR6618 ) .
A cysG mutant ( EB232 ) , whiich requitres cysteine because it lacks siroheme ( 8 ) , did not reduce sulfite to H2S , but did produce H25 from thiosulfate .
An oxrA mutant ( EB216 ) was also tested .
The oxrA gene in S. typhimurium is essential for many anaerobic activities ( 31 ) and appears to be equivalent to the wellknown fnr gene in E. coli ( 14 ) .
EB216 produced very little H2S from thiosulfate , but produced as much as the wild type from sulfite .
A distinction between assimilatory and dissimilatory sulfur reduction was supported by the results of additional tests involving cysteine auxotrophs .
The cysI ( EB218 ) and cysJ ( EB230 ) mutants , which lack the hemoprotein and fiavoprotein components , respectively , of biosynthetic sulfite reductase ( 28 ) , both produced H2S from sulfite and thiosulfate .
Similar ` ly , the cysA ( EB242 ) and cysB ( EB245 ) mutants produced H2S from both compounds .
The , cysB mutants are , reported to be unable to use thiosulfate or sulfite as a sulfur source for aerobic-growth due to the loss of the positive regulator for the cysteine regulon , and cysA mutants are reported to be unable to use thiosulfate due to the lack of the permease for it ( 20 ) .
It is unlikely that the H2S observed in these ` tests was produced from cysteine rather than the inorganiic sulfur compounds because detectable H2S was not produced i n'the absence of thiosulfate or sulfite .
Thiosulfate reductase activity associated with 112S productio ` n .
Earlier studies of thiosulfate reduction by members-of the Enterobacteriaceae ( 10 , 25 , 30 ) used manometric assays of thiosulfate-dependent hydrogen uptake in the presence of hydrogenase from Desulfovibrio spp. .
A more direct assay , in which thiosulfate-dependent methyl viologen oxidation is measured spectrophotometrically , was used by Badziong and Thauer for studies of thiosulfate reduction by D. vulgaris ( 2 ) .
We adapted the latter assay for our studies of S. typhimurium .
The presence of a thiosulfate reductase detected by this assay ( Table 3 ) was found , in most cases , to parallel the results for H2S production .
The results ( Table 3 ) also show that wild-type thiosulfate reductase activity is favored by growth in the absence of air or nitrate and that activity in minimal-medium is dependent on the addition of a sulfur compound .
Thiosulfate was the most effective inducer ; sulfite , sulfide , and cysteine were , respectively , 21 , 60 , and 58 % as effective as thiosulfate .
In nutrient broth , thiosulfate reductase activity was negatively affected by the presence of carbohydrate and only partially de'endent on thiosulfate , probably because the peptone used to formulate nutrient broth contains some thiosulfate in addition to reduced organic sulfur ( 33 ) .
In agreement with tests for H2S production , no thiosulfate reductase was detected in the phs , hemA , and chiA mutants , and reduced levels were found in the oxrA mutant .
Similarily , activities in the H2S-producing cysI , cysJ , and cysG mutants ( data not shown ) did not deviate significantly from the results for the wild type .
The low level of activity in the menB mutant was somewhat surprising , because menquinones would not be expected to participate in methyl viologen-linked thiosulfate reduction .
Wild-type thiosulfate reductase activity was located electrophoretically by means of a methyl viologen-linked activity stain ( Fig. 1 ) .
In many experimnents , a close examination of the activity stain revealed that it was a double band , the lower band being much less pronounced than the major upper band .
Two bands were also observed in an earlier study concerned with chlorate reduction by thiosulfate-reducing enzymes in S. typhimurium ( Riggs and Barrett , Abstr .
Isolation and characterization of inseirtion mutants .
Insertion mutants defective in thiosulfate reduction were isolated by-screening for H2S5 ( white ) colonies on PI agar containing either tetracycline ( for TnlO insertions ) or ampicillin [ for Mu dl ( Apr lac ) operon fusions ] .
Isolates which retained the ability to reduce nitrate and trimethylamnine were kept for further study .
Eight TnlO insertions were obtained ; two were found to be histidine auxotrophs , and in all eight , I -LCB- 2S was linked to the his operon , as is the previous designated phs gene ( 36 ) .
All produced 1125 from sulfite .
Numerous Mu dl ( Apr lac ) H25 mutants were obtained , and Mu dl was immobilized in 21 of them .
Four were histidine auxotrophs Strain Thiosulfate reductase sp act ( U/mg of protein ) Basal mediuma and Supplementsb incubation conditions Carbohydrate Sulfur ( relevant genotype ) Minimal , aerobic Galactose Thiosulfate < 0.02 Minimal + 10 mM Galactose Thiosulfate < 0.02 LT2 ( wild type ) KNO3 , anaerobic Minimal , anaerobic Galactose None < 0.02 Galactose Thiosulfate 2.25 Galactose Sulfite 0.48 Galactose Sulfide 1.35 Galactose Cysteine 1.31 Glucose Thiosulfate < 0.02 Galactose Thiosulfate 1.10 None Thiosulfate 2.15 None None 1.40 CP22 ( phs ) Minimal , anaerobic Galactose Thiosulfate < 0.01 TR6618 ( hemA ) Minimal , anaerobic Galactose Thiosulfate < 0.03 SAST66 ( ubi ) Minimal , anaerobic Galactose Thiosulfate 1.22 TC99 ( menB ) Minimal , anaerobic Galactose Thiosulfate 0.56 TC110 ( chlA ) Minimal , anaerobic Galactose Thiosulfate < 0.03 EB216 ( oxrA ) Minimal , anaerobic Galactose Thiosulfate 0.86 a Minimal medium consisted of VBC formulated in 50 mM phosphate buffer ( pH 6.6 ) , which resulted in a final pH of 6.8 due to the VBC base .
It contained supplements when required by auxotrophs as follows : L-histidine , 0.1 mM ( CP22 ) ; tryptone , 0.5 % ( wt/vol ) ( TR6618 and SAST66 ) ; and 0.1 mM adenosine , 2 mM L-proline , 0.3 mM L-isoleucine , and 0.3 mM L-valine ( TC99 and TC110 ) .
b Concentrations : carbohydrates , 5.6 mM ; Na2S203 , 3 mM ; Na2SO3 , 1 mM ; and L-cysteine , 0.3 mM .
Higher concentrations of Na2SO3 and cysteine could not be used because they inhibited growth .
Nutrient broth , anaerobic All but one could be designated phs , since they produced H2S from sulfite and their mutations exhibited transductional linkage to the his operon ( either to hisD : : TnJO or to zec-2 : : TnJO , which is 76 % linked to hisG46 in strain EB217 .
) The one exceptional strain , EB299 , did not produce H2S from either sulfite or thiosulfate , and its mutation was not linked to his .
It was also found to be incapable of anaerobic-growth-on-glucose minimal-medium and thus did not appear to have a defect related specifically to H2S production .
All of these phs mutants were assayed for thiosulfate reductase and found to be devoid of activity .
The ability of the phs mutants to cross-feed each other was tested in PI agar with all possible combinations of dual inoculation or inoculation of one strain along with the extract from another .
H2S was not produced by any pair of phs mutants .
However , extracts of EB299 did facilitate H2S production by all phs mutants tested .
The latter result suggested that the phs mutants may be defective in the production of an intermediate in thiosulfate reduction .
EB299 , but not any of the phs mutants , produced H2S when combined with extracts of E. coli .
Thus , the phs defect , but not the defect in EB299 , affects an activity that occurs in S. typhimurium but not in E. coli .
Isolation and characterization of temperature-sensitive phs mutants .
Using localized mutagenesis , we isolated a number of phs mutants , seven of which were found to be temperature sensitive for the production of H2S in PI agar ( indistinguishable from the wild type at 30 ' C and H2S at 42 °C .
) Extracts of all seven grown at 30 and 42 °C exhibited reduced thiosulfate reductase activity at 30 °C compared with the wild type and little or no activity at 42 °C .
An extract of one of them ( EB226 ) grown at 30 °C was subjected to electrophoresis and stained for thiosulfate reductase activity .
The activity band exhibited the same relative mobility as the wild-type extract ( Fig. 1 ) .
To see whether the thiosulfate reductase protein produced by any of these mutants during-growth at 30 °C was thermolabile , we assayed extracts heated to 50 °C for 2 h .
The results for three of them are shown in Fig. 2 ; the others all exhibited the same pattern .
Thiosulfate reductase 0 30 60 90 120 Minutes at 50 °C FIG. 2 Stability of thiosulfate reductase ( TSR ) in crude extracts held at 50 °C .
All assays were performed at 30 °C .
Symbols : 0 , LT2 ( wild type ) ; 0 , EB224 ; 0 , EB226 ; A , EB227 aData are given only for phs : : Mu dl mutants which gave detectable activities in anaerobic nutrient broth with or without 3 mM thiosulfate .
b NT , Not tested .
c Other strains were EB234 and EB238 through EB241 .
from the temperature-sensitive mutants was clearly not thermolabile .
These results suggest that phs may not be the structural gene for the thiosulfate reductase detected in our assays .
The phs gene affected in our mutants does , however , encode a protein necessary for thiosulfate reductase activity , as one of the point mutants ( EB244 ) was found to be suppressible by the amber suppressor supD .
Regulation of ( - galactosidase in phs : : Mu dl ( lac ) fusion mutants .
Nine of the 20 phs : : Mu dl operon fusion mutants synthesized , B-galactosidase under anaerobic-growth-conditions with and without thiosulfate ( Table 4 ) .
Surprisingly , the activities of these strains were very low in the minimal-medium which had yielded optimal wild-type thiosulfate reductase activity ( Table 3 .
) Aliabadi et al. ( 1 ) also noted a requirement for complex medium in the expression of Igalactosidase by phs : : Mu dl mutants .
We examined many different variables to determine which organic or inorganic components of nutrient broth might be required for induction of expression in minimal-medium .
Three types of regulatory factors were identified : carbon source , reduced sulfur available , and growth stage .
The effects of carbon source and reduced sulfur are summarized in Table 5 .
The repression by glucose was no surprise , because glucose had already been found to interfere with wild-type H2S production ( Table 2 ) and thiosulfate reductase activity ( Table 3 ) .
More surprising was the finding that galactose had a similar but less severe effect .
Far higher activities were obtained with glycerol and fumarate as the carbon and energy sources .
High activities were also obtained with glucose 1-phosphate ( data not shown ) .
Sulfur compounds other than thiosulfate were found to be essential for significant 3-galactosidase activity in minimal-medium .
P-Galactosidase activity in the presence of reduced glutathione or sodium sulfide was as high as that obtained with nutrient broth added to the minimal-medium , suggesting that reduced sulfur in the nutrient broth may have been a key factor in the induction of expression by complex medium .
The effect of growth stage on 3-galactosidase activity in EB222 ( Fig. 3 ) was quite significant .
In the minimal-medium , a sharp peak in activity was observed during the exponential phase , possibly at an inflection point in the growth-rate .
In nutrient broth , the activity rose until the late exponential phase , but did rnot decline in the stationary-phase .
The OD650 at which the P-galactosidase peak occurred varied with the composition of the medium .
Thus , some of the variation in 3-galactosidase activity from carbon source to carbon source obtained in the previous experiments when cells were assayed at about the same OD650 might actually be explained by differences in the relationship between the timing of the assay and the timing of the peak .
On the other hand , a comparison of peak heights obtained with galactose ( Fig. 3A ) and glycerol-fumarate ( Fig. 3B ) shows that the highest activity obtained with galactose was far lower than that obtained with glycerol-fumarate .
Regulation of phs by oxrA .
To see whether the lowered levels of H2S production and thiosulfate reductase activity in the oxrA mutant were due to a need for the oxrA product for full expression of phs , we moved an oxrA : : TnJO insertion into the phs : : Mu d fusion mutant and assayed P-galacto-sidase activity in nutrient broth .
The activities obtained were KNO3 , anaerobic Minimal , anaerobic Galactose None < 0.02 Galactose Thiosulfate 2.25 Galactose Sulfite 0.48 Galactose Sulfide 1.35 Galactose Cysteine 1.31 Glucose Thiosulfate < 0.02 Galactose Thiosulfate 1.10 None Thiosulfate 2.15 None None 1.40 CP22 ( phs ) Minimal , anaerobic Galactose Thiosulfate < 0.01 TR6618 ( hemA ) Minimal , anaerobic Galactose Thiosulfate < 0.03 SAST66 ( ubi ) Minimal , anaerobic Galactose Thiosulfate 1.22 TC99 ( menB ) Minimal , anaerobic Galactose Thiosulfate 0.56 TC110 ( chlA ) Minimal , anaerobic Galactose Thiosulfate < 0.03 EB216 ( oxrA ) Minimal , anaerobic Galactose Thiosulfate 0.86 a Minimal medium consisted of VBC formulated in 50 mM phosphate buffer ( pH 6.6 ) , which resulted in a final pH of 6.8 due to the VBC base .
It contained supplements when required by auxotrophs as follows : L-histidine , 0.1 mM ( CP22 ) ; tryptone , 0.5 % ( wt/vol ) ( TR6618 and SAST66 ) ; and 0.1 mM adenosine , 2 mM L-proline , 0.3 mM L-isoleucine , and 0.3 mM L-valine ( TC99 and TC110 ) .
b Concentrations : carbohydrates , 5.6 mM ; Na2S203 , 3 mM ; Na2SO3 , 1 mM ; and L-cysteine , 0.3 mM .
Higher concentrations of Na2SO3 and cysteine could not be used because they inhibited growth .
C o , 1.00-so a : 0.15 .
Thiosulfate reductase in cell extract preparations subjected to electrophoresis in a 6 % polyacrylamide gel .
Lanes : A , temperature-sensitive mutant EB226 grown at 30 °C ; B , phs : : Mu dl mutant EB3222 ; C , wild-type LT2 .
P-Galactosidase activity in the phs : : Mu dl ( lac ) operon fusion mutantsa 1-Galactosidase activity ( u/OD60 , unit ) Aerobic Anaerobic-growth Strain growth ; Nutrient broth Galactose nutrient broth with Without With VBC minimal thiosulfate thiosulfate thiosulfate with thiosulfate EB22 EB233 30 15 556 518 620 472 77 33 EB236 and EB237 All othersc < 1 7-25 26-28-257-327 26-28-241-308 NTb 23-38 TABLE 5 .
Effect of carbon source and reduced sulfur on,-galactosidase expression in strain EB222 Supplementsa,-Galactosidase activity Basal Sulfur medium Carbon ( U/OD600 unit ) None None 556 None Thiosulfate 620 Nutrient broth None Sulfite 891 None Sulfide 383b Galactose None 170 Glucose None 53 Glycerol-fumarate None 488 Glycerol-fumarate None 38 Glycerol-fumarate Thiosulfate 116 Glycerol-fumarate Sulfite 272 Glycerol-fumarate Sulfide 339 Glycerol-fumarate Cysteine 281 Glycerol-fumarate Glutathione ( reduced ) 367 Glycerol-fumarate Glutathione ( oxidized ) 97 Glycerol-fumarate Nutrient broth 377 Galactose Sulfide 146 Glucose Sulfide 35 Glycerol-fumarate None 24 Glycerol-fumarate Sulfide 93 a Supplement concentrations : carbon compounds , 10 mM ; inorganic sulfur compounds , 1 mM ; organic sulfur compounds , 0.5 mM ; nutrient broth , 0.8 % .
b Extremely poor growth Minimal ( M9 ) Minimal ( VBC ) 0.500 : ¬ -- -900 * 800 a. 700 4 .
500 -- 400 0 p. 10oo 0 0 0.010 , .30 2000 100 I-u 14 D f I -- 1 I 4 6 8 10 Incubation time ( hr ) 2 i .
2 0 1 0.500 I CC -- I * 1000 .900 Zo a 800 a. .700 600 0 .
O10Q ( 0.001 0 I 4 6 8 10 1 1 2 2 Incubation time ( hr ) FIG. 3 .
Timing of phs expression in phs : : Mu dl mutant EB222 .
( A ) Galactose minimal ( M9 ) medium ; ( B ) glycerol-fumarate minimal ( M9 medium ; ( C ) nutrient broth ( unsupplemented ) .
Symbols : 0,0 , growth as measured by OD650 in a Bausch and Lomb spectrophotometer ; O , U , , B-galactosidase activity ; 0,0 , no sulfide added ; * , E , 1 mM Na2S added to growth medium .
actually slightly higher than those obtained for the Oxr + strain .
The ` same oxrA : : TnlO insertion has been shown to abolish 13-galactosidase activity in a chiC : : Mu dl strain ( 19 ) .
Apparently , phs , although anaerobically induced ( Table 4 ) , is not under the global regulation of oxrA .
DISCUSSION The results presented here show that the pathways in S. typhimurium for the anaerobic reduction of thiosulfate and sulfite to 112S are distinct both from each other and from the analogous reductions of the cysteine biosynthetic pathway .
112S production from thiosulfate was shown to be associated with an anaerobically induced thiosulfate redujtase that catalyzed the reduction of thiosulfate with methyl viologen .
Benzyl viologen could not serve as the electron-donor because the Eo ' for the S2032/032 + S2-redox couple ( ' -400 mV ) ( 32 ) is lower than that for benzyl viologen ( -360 mV ) .
Thiosulfate reductase was absent in a heme mutant and in a chlA mutant , which suggests that it may be a hemecontaining or cytochrome-associated molybdoenzyrne .
As noted previously ( 16 , 18 ) , H2S was not produced from thiosulfate by menaquinone mutants , indicating a role for a menaquinone in electron transfer to thiosulfate .
In contrast , anaerobic reduction of sulfite to H2S did not require menaquinones or a molybdoenzyme .
Sulfite reduction did appear to require siroheme , which is also necessary for anaerobic nitrite reduction as well as biosynthetic sulfite reduction in the Enterobacteriaceae ( 8 ) .
The presence of this anaerobic sulfite reductase may explain why cysl and cysJ mutants , which are defective in biosynthetic sulfite reductase and require cysteine for aerobic-growth , can grow without cysteine anaerobically ( 3 ) .
We isolated a number of hydroxylamine , TnlO insertion , and Mu dl operon fusion mutants which could be designated phs mutuants on the basis of phenotype and mutation location .
All insertion and fusion mutants were devoid of thiosulfate-dependent methyl viologen oxidation activity .
Several of the point mutants were temperature sensitive for H2S production as well as thiosulfate reductase activity , but none of them produced a thermolabile enzyme when grown at the permissive temperature , and the enzyme in the strain examined electrophoretically exhibited the same relative mobility as the wild-type enzyme .
These results suggested that phs is not the structural gene for methyl viologen-linked thiosulfate reductase .
We had noted that in many electro-phoretic experiments , the activity stain appeared to be a double band .
If there are actually two thiosulfate reductases , then our mutant selection procedure was biased towards the selection of regulatory mutants , because two structual gene mutations would be required to produce the H2S phenotype .
Another possible explanation for the lack of a thermolabile in the enzyme temperature-sensitive mutants is that the methyl viologen-linked reductase detected in our assays and in electrophoresis was only part of the activity encoded by the phs gene .
Use of an assay in which H2S from thiosulfate is quantified would help to answer that question .
Our initial assays of P-galactosidase expression by the phs : : Mu dl ( lac ) mutants resulted in much higher activities in nutrient broth than in the minimal-medium used for wild-type thiosulfate reductase assays .
Subsequent experiments revealed that higher levels of expression in the minimal me-dium could be obtained by changing the carbon source , adding reduced sulfur , and adjusting the timing of the assay relative to the growth stage of the culture .
A possible explanation for the fact that reduced sulfur was required for significant P-galactosidase expression in the phs : : Mu dl operon fusion mutant while thiosulfate was the most effective inducer of wild-type thiosulfate reductase is that the true inducer of phs is some form of reduced sulfur which is synthesized from thiosulfate by the action of the phs gene .
The Phs-strains would be unable to synthesize this compound .
The finding that menaquinone mutants , which can not reduce thiosulfate to H2S , contained only 25 % of the wildtype methyl viologen-linked thiosulfate reductase is consistent with a role for a product of thiosulfate reduction in the induction of the pathway .
The severe repression exerted by glucose is suggestive of catabolite-repression , which has not been shown to be an important regulatory factor for the major nitrate reductase ( 5 ) or trimethylamine oxide reductase ( 17 ) .
Preliminary studies of this aspect of phs regulation ( M. A. Clark and E. L. Barrett , Curr .
in press ) showed that many sugars were capable of repressing phs expression , including some that are themselves subject to catabolite-repression , e.g. , L arabinose and L-rhamnose .
Further studies of this complex aspect of phs regulation are now in progress .
2 These studies supported by Public Health Service were grant AI-22685 from the National Institutes of Health and by funds from the California Agricultural Experiment Station .
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