Effects of Anaerobic Regulatory Mutations and Catabolite Repression on Regulation of Hydrogen Metabolism and Hydrogenase Isoenzyme Composition in Salmonella typhimurium DEREK J. JAMIESON , R. GARY SAWERS , PAUL A. RUGMAN , DAVID H. BOXER , AND CHRISTOPHER F. HIGGINS * Department of Biochemistry , Medical Sciences Institute , University of Dundee , Dundee DDI 4HN , Scotland Hydrogen metabolism in Salmonella typhimurium is differentially regulated by mutations in the two anaerobic regulatory pathways , defined by the fnr ( oxrA ) and oxrC genes , and is controlled by catabolite-repression .
The synthesis of the individual hydrogenase isoenzymes is also specifically influenced by fnr and oxrC mutations and by catabolite-repression in a manner entirely consistent with the proposed role for each isoenzyme in hydrogen metabolism .
Synthesis of hydrogenase isoenzyme 2 was found to befnr dependent and oxrC independent , consistent with a role in respiration-linked hydrogen uptake which was shown to be similarly regulated .
Also in keeping with such a respiratory role was the finding that both hydrogen uptake and the expression of isoenzyme 2 are under catabolite-repression .
In contrast , formate hydrogenlyase-dependent hydrogen evolution , characteristic of fermentative growth , was reduced in oxrC strains but not infnr strains .
Hydrogenase 3 activity was similarly regulated , consistent with a role in hydrogen evolution .
Unlike the expression of hydrogenases 2 and 3 , hydrogenase 1 expression was both fnr and oxrC dependent .
Hydrogen uptake during fermentative growth was also both fnr and oxrC dependent .
This provided good evidence for a distinction between hydrogen uptake during fermentation-and respiration-dependent growth and for a hydrogen-recycling process .
The pattern of anaerobic control of hydrogenase activities illustrated the functional diversity of the isoenzymes and , in addition , the physiological distinction between the two anaerobic regulatory pathways , anaerobic respiratory genes being fnr dependent and enzymes required during fermentative growth being oxrC dependent .
Escherichia coli and Salmonella typhimurium each possess at least three distinct hydrogenase isoenzymes ( 22 , 24 ) , which together are responsible for both hydrogen uptake and hydrogen-evolving activities .
The hydrogen uptake ( respiratory ) activity is involved in anaerobic energy generation ( 1 , 8 ) , whereas the hydrogen-evolving reaction is catalyzed by the formate hydrogenlyase system ( 20 ) .
Two membrane-bound hydrogenases , isoenzymes 1 and 2 , have been purified from E. coli and characterized as nickel-containing metalloenzymes ( 2 , 3 , 23 ) .
S. typhimurium also contains functional equivalents to isoenzymes 1 and 2 which are immu-nologically related to their E. coli counterparts ( 24 ) .
A third hydrogenase activity , immunologically distinct from isoenzymes 1 and 2 , is also present in membranes from anaerobically grown E. coli and S. typhimurium ( 23 , 24 ) .
Previously ( 24 ) , we identified distinct physiological roles for these three isoenzymes .
Isoenzymes 1 and 2 catalyze hydrogen uptake reactions ; isoenzyme 1 functions during fermentative growth , whereas isoenzyme 2 is active only during respiration-dependent growth .
Hydrogenase 3 catalyzes hydrogen evolution and appears to form part of the formate hydrogenlyase pathway .
Hydrogen is metabolized by enterobacteria only during anaerobic-growth .
We have identified two genetically and physiologically distinct regulatory pathways for the anaerobic control of gene expression ( 7 ) .
The fnr ( 25 ) ( designated oxrA in S. typhimurium [ 28 ] ) and oxrC genes are two pleiotropic regulatory loci which define two distinct classes of anaerobically induced genes .
We have suggested that fnr may control the expression of anaerobic respiratory genes ( 5 , 9 , 10 , 16 , 27 ) , whereas oxrC principally regulates the synthesis of enzymes with fermentative or biosynthetic roles ( 7 ) .
Because the hydrogenases serve both respiratory and fermentative functions ( 24 ) , we examined the effects of fnr and oxrC mutations on hydrogen metabolism and on the synthesis of the individual hydrogenase isoenzymes .
Each of the hydrogenase isoenzymes was shown to be subject to distinct control processes , which provides strong support for the physiological roles proposed for the enzymes ( 24 ) and for the distinct physiological roles of thefnr and oxrC regulatory pathways .
MATERIALS AND METHODS Bacterial strains .
The bacterial strains used in this study as well as their genotypes and construction are detailed in Table 1 .
Cells were grown anaerobically in nutrient broth ( NB ; Difco Laboratories ) at 37 °C unless otherwise stated .
Anaerobic-growth was without shaking in completely filled screw-cap vessels , or , alternatively , GasPaks ( Oxoid Ltd. ) were used to provide an anaerobic environment .
LB-medium was described by Miller ( 14 ) .
NB was supplemented when appropriate with additional carbon sources at 0.4 % or with other additives as follows : sodium nitrate , 10 g liter - ' ; sodium formate , 0.5 g liter - ' ; and sodium fumarate , 5 g liter - ' .
Ammonium molybdate and K2SeO3 were added to all growth media at 1 puM .
Kanamycin and ampicillin were used at 50 , ug ml-1 .
MacConkey agar-nitrate and glycerol-nitrate plates were as described by Barrett et al. ( 4 ) and Lambden and Guest ( 10 ) , respectively .
Transductions were performed by 40 E. coli P4X Hfr metBi E. Wollman a All typhimurium strains are derivatives of LT2 ( Z ) except for CH602 S. and CH805 , which are LT2 ( A ) derivatives .
boxrA is equivalent to the fnr locus of E. coli ( 7 , 28 ) .
The Mu dl ( Ap lac ) fusions in CH953 and CH975 are stabilized .
using a high-transducing derivative of P22 int4 .
fnr ( oxrA ) mutations were moved into appropriate recipients by transduction to Kanr with a P22 lysate of CH602 ( zda : : TnS ; 70 % linked to fnr [ oxrA ] [ 7 , 28 ] ) .
Cotransfer of the fnr allele with the TnS was determined by screening Kanr transducfor the inability anaerobically on glycerol-tants to grow nitrate and for their growth small , deep red colonies plates as on anaerobic MacConkey agar-nitrate plates .
The oxrC mutation was moved between strains by transduction to Kanr with a P22 lysate of CH805 ( oxrCJ02 : : TnS ) .
Transductants were shown to have acquired the OxrC phenotype by screening for white colonies MacConkey agar-glucose on plates ( 7 ) .
The cya : : TnJO and crp : : TnJO mutations were transferred from strains PP1002 and PP1037 ( P. W. Postma , Amsterdam ) , respectively , by transduction to BCP Institute , Tetr with P22 lysates of these strains .
The acquisition of cya and confirmed by the loss of the ability to grow crp was anaerobically on minimal-medium-glycerol plates .
After any transductions involving transposable elements , the correct location of the transposon was checked genetically by marker rescue .
All enzyme assays were as described previously ( 24 ) .
Hydrogenase activity was measured as the hydrogen-dependent reduction of benzyl viologen .
A unit of activity is 1 , umol of benzyl viologen reduced per min .
Hydrogen uptake was measured as fuma-rate-dependent hydrogen uptake .
A unit represents 1 , umol of hydrogen oxidized per min .
Formate hydrogenlyase activity was monitored as the formate-dependent evolution of hydrogen .
A unit represents 1 , umol of hydrogen evolved per min .
A unit of formate dehydrogenase ( FDH-BV ) activity is 1 p.g ion of formate oxidized per min ( 24 ) .
A unit of fumarate reductase activity represents 1 , umol of fumarate reduced per min ( 24 ) .
Nitrate reductase was assayed as the nitrate-dependent oxidation of reduced benzyl viologen ( 15 ) ; a unit of activity is 1 , ug ion of nitrate reduced per min .
, - Galactosidase was assayed as described by Miller ( 14 ) .
Protein was estimated by the method of Lowry et al. ( 12 ) .
Antibodies to E. coli hydrogenase isoenzymes 1 and 2 were raised in rabbits , and the immunoglobulin fractions were purified as described previously ( 2 , 23 ) .
Rocket immunoelectrophoresis was performed as described previously ( 6 ) .
Immunological quantitation of the hydrogenase activity in Triton X-100-dispersed membranes , which is associated with either isoenzyme 1 or isoenzyme 2 , was as described previously ( 24 ) .
The remaining activity , not precipitable with antibodies against either isoenzyme 1 or 2 , is termed hydrogenase 3 and was also estimated as described previously ( 24 ) .
Activities of the individual hydrogenase isoenzymes in Triton X-100-dispersed membranes are expressed as micromoles of benzyl viologen reduced per minute per milligram of protein .
Source Straina S. typhimurium LT2 ( Z ) CH602 B. N. Ames 7 , 28 Wild type pepl7 : : Mu dl oxrAl zda-893 : : Tn5 AoppBC250 tppB84 : : Mu dl-8 oxrCJ02 : : Tn5 hyd : : Mu dl ( Ap lac ) oxrC102 : : Tn5 hyd : : Mu dl ( Ap lac ) oxrAl zda-893 : : Tn5 oxrAl zda-893 : : TnS oxrC102 : : Tn5 cya : : TnlO crp : : TnlO 7 CH805 CH953 7 RESULTS Because LT2 ( Z ) is the only wild-type isolate of S. typhimurium which has all three hydrogenase isoenzymes ( 24 ) , all studies were performed with isogenic derivatives of this strain unless otherwise indicated .
Mutations in oxrC and fnr ( oxrA ) were transduced into LT2 ( Z ) as described in Materials and Methods .
When total hydrogenase activity ( H2 : benzyl viologen oxidoreductase ) was examined , neither fnr nor oxrC mutations had major effects ( Table 2 ) .
However , when hydrogenase activity was separated into its component parts , specific effects of these mutations could be demonstrated .
Regulation of formate hydrogenlyase activity .
Formate activity ( hydrogen evolution ) was present in hydrogenlyase cells fermentatively ( glucose ) and was further induced grown However , activity was essenby added formate ( Table 2 ) .
tially absent during respiration-dependent growth ( glycerolformate medium ) .
The oxrC and fnr mutations had very different effects on formate hydrogenlyase activity .
Muta-in oxrC resulted in a large reduction in activity and , tions although activity could be restored to some extent by the addition of formate , it never reached wild-type exogenous levels .
This that formate hydrogenlyase is induced implies anaerobiosis and the oxrC mutation both by by formate ; prevents anaerobic induction but has little effect on induc-of the fhl gene is tion by exogenous formate .
Unlike oxrC mutations , mutations in caused a reduction in formate hydrogenlyase fnr only slight activity which was restored to normal levels by formate .
We have a although somewhat more marked , reported similar , effect in E. coli ( 22 ) .
It seems likely that fnr does not affect a Cells were harvested at the late exponential phase of growth , washed once , resuspended in 100 mM potassium phosphate ( pH 6.8 ) , and immediately assayed for the activities listed above .
b Shown are specific activities for whole-cells .
For definitions of units of activity , see Materials and Methods the anaerobic induction of formate hydrogenlyase directly but that the effect is an indirect consequence of a reduction of endogenous formate synthesis , presumably brought about by a reduction of pyruvate formate lyase activity ( 22 ) .
Thus , the anaerobic induction of formate hydrogenlyase activity is oxrC dependent and fnr independent .
Regulation of hydrogen uptake .
Previously ( 24 ) , we presented evidence which suggests that E. coli and S. typhimurium possess two distinct hydrogen uptake activities catalyzed by different isoenzymes , one functioning during fermentative growth and the other functioning during respi-ration-dependent growth .
An examination of the effects of oxrC and fnr mutations on hydrogen uptake strongly supported this conclusion .
Mutations in oxrC had no effect on hydrogen uptake during respiration-dependent growth ( glyc-erol-fumarate medium ; Table 2 ) .
However , hydrogen uptake in cells grown fermentatively , with or without formate , was completely abolished .
This is in contrast to the effects offnr mutations , which abolish hydrogen uptake under all growth-conditions .
Thus , the oxrC mutation provides a clear distinction between the two modes of hydrogen uptake .
Effects of oxrC andfnr mutations on hydrogenase isoenzyme content .
There are at least three distinct hydrogenase isoenzymes in E. coli and S. typhimurium ( 24 ) .
Because of the differential effects of oxrC and fnr mutations on the different modes of hydrogen metabolism , it was important to assess the effects of these mutations on each individual hydrogenase isoenzyme ( Table 3 ) .
Mutation s in oxrC dramatically reduced isoenzyme 1 activity , even iin the presence of endogenous formate which , in the wild-type strain , was an a Cells were harvested , and the membrane fractions were prepared as described in Materials and Methods .
b Expressed per milligram of protein in the Triton X-100-dispersed membrane fractions .
1 and 2 Isoenzyme activities were calculated from the activities immunoprecipitated with antibodies specific for isoenzyme 1 or 2 , respectively .
The nonimmunoprecipitable activity was that activity not immunoprecipitated by a mixture of antibodies specific for isoenzymes 1 and 2 .
inducer of isoenzyme 1 expression .
Conversely , oxrC mutations had no effect on isoenzyme 2 activity and did not impair the characteristic enhancement of isoenzyme 2 during respiratory growth ( glycerol-fumarate medium ) .
The effects of oxrC mutations are distinct from those of fnr mutations , which reduce both hydrogenase 1 and 2 activities ( Table 2 ) .
The presence of hydrogenase 1 and 2 antigens in the oxrC andfnr mutants was also analyzed immunologically ( Fig. 1 ) .
In all cases , the presence or absence of antigens corresponded with the differences in isoenzyme activity .
This implies that the regulation of hydrogenase isoenzyme composition is at the level of synthesis ( transcription/translation ) rather than by modulation of enzyme activity .
The hydrogenase activity not associated with isoenzymes 1 and 2 ( hydrogenase 3 ) was somewhat reduced by the oxrC mutation , and the formate induction of this activity was also impaired .
In contrast , fnr mutants had essentially normal and fully formate-inducible hydrogenase 3 activity , although , in the absence of formate , fnr mutations did result in a slight reduction in hydrogenase 3 activity .
This has also been observed for E. coli ( 22 ) and is probably an indirect effect caused by reduced synthesis of endogenous formate in the fnr mutant .
Mutations in the tppR gene , which affect a subset of oxrC-dependent genes ( 7 ) , had no effect on cellular hydrogen metabolism or on the synthesis of the individual hydrogenase isoenzymes ( data not shown ) .
The above results , together with the effects of oxrC and fnr mutations on hydrogen metabolism ( see above ) , are entirely consistent with the proposed role for each of the individual isoenzymes .
Hydrogenase 2 is believed to catalyze respiration-dependent hydrogen uptake ( 24 ) .
In keeping with this , fnr mutants , but not oxrC mutants , were found to have reduced respiration-dependent hydrogen uptake and reduced 2 hydrogenase activity .
Similarly , hydrog-enase 1 is believed to participate in hydrogen uptake under fermentative growth-conditions .
The reduction in this activity by fnr and oxrC mutations can be entirely explained by their similar effects on the cellular content of isoenzyme 1 .
Finally , the reduction in formate hydrogenlyase activit 7 CH975 CH1019 CH1021 CH1107 CH1108 7 7 7 This study This study TABLE 2 .
Effect of mutations in the anaerobic regulatory genes oxrC and on hydrogen metabolism fnr ( oxrA ) Sp act ( U/mg of protein ) oft : Formate Hydrogen Hydrogenase hydrogenlyase uptake Growth conditions and strainsa Glucose LT2 ( Z ) 0.449 0.103 0.129 0.412 0.320 0.054 0.146 < 0.001 < 0.001 CH1019 ( fnr ) CH1021 ( oxrC ) Glucose + formate LT2 ( Z ) CH1019 ( fnr ) CH1021 ( oxrC ) 0.699 0.592 0.277 0.475 0.260 0.711 0.091 < 0.001 < 0.001 Glycerol + fumarate LT2 ( Z ) CH1019 ( fnr ) CH1021 ( oxrC ) 0.164 0.022 0.377 0.049 0.059 0.039 0.327 < 0.001 0.367 VOL .
168 , 1986 REGULATION OF HYDROGEN METABOLISM TABLE 3 .
Effect of mutations in the anaerobic regulatory genes oxrC and fnr ( oxrA ) on hydrogenase isoenzyme content Hydrogenase sp act ( U/mg of protein ) ofb : Isoenzyme Isoenzyme preipitab .
1 2 precipitable activity Growth conditions and strainsa Glucose 0.115 0.655 0.187 0.806 0.315 0.255 0.272 LT2 ( Z ) ( wild type ) CH1019 ( fnr ) CH1021 ( oxrC ) < 0.005 < 0.005 Glucose + formate LT2 ( Z ) ( wild type ) CH1019 ( fnr ) CH1021 ( oxrC ) 0.097 0.332 0.077 0.726 1.980 1.817 < 0.005 < 0.012 0.569 Glycerol + fumarate LT2 ( Z ) ( wild type ) CH1019 ( fnr ) CH1021 ( oxrC ) < 0.005 1.25 0.684 < 0.005 0.242 0.827 < 0.005 1.897 0.452 A A p A n * b. h 1 * l f * b c d B A A ) 0 ) ^ A 0 0 a h i j b ` C-0 a f FIG. 1 .
Hydrogenase isoenzyme 1 and 2 antiigen contents of strains carrying lesions in the oxrC and fnr ( oxrO4 ) genes .
Triton X-100-dispersed membrane fractions ( 40 , ug of pr ( otein ) were analyzed by rocket immunoelectrophoresis with antit , odies to E. coli hydrogenase isoenzyme 1 ( A ) or E. coli hydrogen ; ase isoenzyme 2 ( B ) .
Lanes : LT2 ( Z ) with glucose ; b , a , grown as CH1019 ( fnr ) ; for lane but CH1021 ( oxrC ) ; di fLT2 ( ne ) abwunt c , as a , with glucose plus formate ; e , as for lane d , but CH1 ( ) 19 ( fnr ) ; f , as for lane d , but CH1021 ( oxrC ) ; g , LT2 ( Z ) grown wiith glycerol plus fumarate ; h , as for lane g , but CH1019 ( fnr ) ; i , as for lane g , but CH1021 ( oxrC ) ; j , E. coli P4X grown with glucose plus formate .
Sp act ( U/mg of protein ) of : Fumarate reductase aRie Str elnvant genotype Strain Nitrate reductase LT2 ( Z ) Wild type 3.24 0.11 ( 0.16 ) 3.31 2.35 CH1019 fnr 0.04 0.04 ( 0.17 ) 1.37 0.14 CH1021 oxrC 3.68 0.026 ( 0.06 ) 1.17 4.21 a Cells were grown anaerobically in NB supplemented-with-glucose ( and KNO3 when nitrate reductase was assayed ) , and the membrane fractions were prepared as described in Materials and Methods .
All assays were performed on the membrane fraction except for that for FDH-BV for which intact cells were used .
For definitions of units of activity , see Materials and Methods .
b Values in parentheses refer to FDH-BV activity in whole-cells grown in NB supplemented-with-glucose and sodium formate .
caused by mutations in oxrC , but not by mutations in fnr , parallels the effects of these enzymes on hydrogenase 3 activity , in agreement with the proposed participation of hydrogenase 3 in hydrogen evolution .
Regulation of formate dehydrogenase activity .
Because formate hydrogenlyase requires the function of FDH-BV , as well as a hydrogenase , it was necessary to examine the effects of oxrC and fnr mutations on this activity .
In complete agreement with its minor effect on formate hydrogenlyase activity , an fnr mutation only resulted in a small reduction in FDH-BV activity and this could be restored to normal levels by the addition of formate ( Table 4 ) .
The effects of an oxrC mutation were , however , more pronounced .
Thus , oxrC mutations reduced FDH-BV levels , and activity could not be restored by formate .
This is again consistent with the effects of oxrC on formate hydrogenlyase activity .
Indeed , it seems that the effects of the oxrC mutation on FDH-BV activity are more important in determining hydrogen evolution than are its effects on hydrogenase 3 activity since substantial hydrogenase 3 activity was detected under all conditions examined .
However , it should be remembered that hydrogenase 3 activity may be due to two or more isoenzymes , only one of which is associated with formate hydrogenlyase .
The effects of the oxrC and fnr mutations on FDH-BV activity are very similar to their effects on the expression of an ffil-lacZ fusion ( 4 , 7 ) .
The flhl-acZ fusion used in these experimeents lacked formate hydrogenlyase activity but still retained hydrogenase activity .
Thus , the fusion was believed to be a fusion to the structural gene for FDH-BV .
However , in the light of our findings that only one of the hydrogenase isoenzymes is required for formate hydrogenlyase activity , it was important to establish the isoenzyme contents of strains carrying this fhl-lacZ fusion .
Direct immunoprecipitation showed that all three hydrogenase isoenzymes were present in normal amounts ( data not shown ) .
This is consistent with flhl being the structural locus for FDH-BV ( 4 , 17 , 18 ) , and it also eliminates the possibility that the effects of oxrC andfnr mutations on the hydrogenase isoenzymes ar.-amdiated via the fhi locus .
The tppR mutation ( 7 ) had no effect on FDH-BV activity , as anticipated from its lack of effect onjhl expression ( 7 ) .
In addition to FDH-BV , the effects of oxrC and fnr mutations on other anaerobically induced enzymes were also examined ( Table 4 ) .
Neither nitrate reductase nor fumarate reductase activity was affected by an oxrC mutation .
However , as anticipated from results with E. coli ( 10 , 16 ) , both activities were drastically reduced in fnr strains .
Phenotypic suppression of the oxrC mutation .
We have shown that the oxrC mutation can be phenotypically suppressed by growth on fructose ( 7 ) .
Normal hydrogen metab-olism was found for an oxrC strain after growth on fructose rather than on glucose ( Table 5 ) .
Both hydrogen uptake during fermentative growth and isoenzymne 1 levels were restored to wild-type levels .
Similarly , hydrogen evolution and the activities of the enzymes of the formate hydrogen-lyase pathway ( hydrogenase 3 and FDH-BV ) were also fully restored by growth on fructose .
Hydrogen metabolism and catabolite-repression .
Since the hydrogenase isoenzymes were differentially expressed during fermentative and nonfermentative growth , it seemed likely that expression of the isoenzymes might be controlled by catabolite-repression .
Strains were constructed carrying cya and crp mutations , and hydrogenase activities were determined for cells grown fermentatively ( glucose ) in the presence or absence of added cyclic AMP ( cAMP ) ( Table 6 ) .
Two major effects were observed .
First , hydrogenase isoenzyme 2 was found to be subject to catabolite-repression .
The cellular content of this isoenzyme was enhanced by the addition of cAMP , both in the wild-type and in the cya strain ( Fig. 2c and d ) .
This enhancement was not seen in the crp strain ( Fig. 2e and f ) , showing the cAMP effect to be mediated by the cAMP-receptor-protein ( CRP ) .
However , it should be noted that there was always a substantial basal level of isoenzyme 2 expression which was maintained independently of the catabolite-repression system .
The repression of isoenzyme 2 during fermentative ( glucose ) growth is , of course , compatible with the respiratory role of this enzyme .
The second important effect was that added cAMP was found to reduce formate hydrogenlyase activity , particularly in a cya mutant .
Curiously , the addition of cAMP to the growth medium of wild-type cells did not fully mimic the effects seen for the cya mutant ; we have no explanation for this at present .
Again , the cAMP effects were dependent upon CRP , since cAMP had no effect in a crp strain .
The effects of cAMP on hydrogenase isoenzyme 3 paralleled its effects on formate hydrogenlyase activity , consistent with this isoenzyme being associated with the formate hydrogen-lyase pathway and with its fermentative role .
As for isoenzyme 3 , the levels of isoenzyme 1 were also reduced by the addition of cAMP ( Fig. 2a and b ) .
However , cya and crp mutations had complex effects on the expression of this isoenzyme , which are not fully understood at present .
Thus , it is clear that expression of the individual hydrogenase isoenzymes is regulated by the cAMP-CRP system in a manner consistent with their fermentative or nonfermentative roles .
Suppression of the oxrC effect on hydrogen metabolism by fermentative growth on fructose Sp act ( U/mg of protein ) of : Strain and growth-conditionsa Hydrogenasec Isoenzyme 2 Formate Hydrogen uptakeb hydrogenlyaseb Nonimmuaoprecipitable activity 0.546 0.703 0.272 1.01 Isoenzyme 1 LT2 ( Z ) with glucose LT2 ( Z ) with fructose 0.811 0.968 1.05 0.706 0.079 0.046 0.008 0.123 0.042 0.035 0.002 0.052 0.207 0.183 0.067 0.282 CH1021 ( oxrC ) with glucose CH1021 ( oxrC ) with fructose a Cells were harvested at the late exponential phase of growth , washed once , and resuspended in 100 mM potassium phosphate ( pH 6.8 ) .
The assays on the whole-cells were performed immediately .
Membrane fractions were prepared as described in Materials and Methods .
b Assays performed on whole-cells .
c Expressed per milligram of protein in the Triton X-100-dispersed membrane fractions .
Isoenzyme 1 , isoenzyme 2 , and nonimmunoprecipitable hydrogenase activities were calculated as described in Materials and Methods and in Table 3 - A - a bcdef FIG. 2 .
Hydrogenase isoenzyme 1 and 2 antigen contents of strains defective in catabolite-repression .
Triton X-100-dispersed membrane fractions ( 40 p.wg of protein ) were analyzed by rocket immunoelectrpphoresis on a divided immunoplate using antibodies to E. ccli hydrogenase isoenzyme 2 ( top layer ) and to E. ccli hydrogenase isoenzyme 1 ( bottom layer ) .
All strains were grown anaerobica , lly in NB containing glucose and cAMP ( 5 mM ) as indicated .
Samples are from the same experiment described in Table 6 .
Lanes , a , LT2 ( Z ) ; b , as for lane a , but grown with cAMP ; c , CH11O7 ( cya ) ; d , as for lane c , but grown with cAMP ; e , CHliO8 ( crp ) ; f , as for lane e , but grown with cAMP .
DISCUSSION In this paper we describe in detail the regulation of hydrogen metabolism in S. typhimurium , both by anaerobic regulatory mutations and in response to different growth-conditions .
An examination of the specific regulation of each of the individual hydrogenase isoenzymes has provided strong support for the proposed functions of each of the isoenzymes in hydrogen metabolism and , in addition , defines the physiologically distinct nature of the oxrC-and fnr-dependent anaerobic regulatory pathways .
The oxrC and fnr mutations define pleiotropic anaerobic regulatory pathways , which we have suggested may serve distinct physiological roles ( 7 ) .
Because the hydrogenase isoenzymes serve both fermentative and respiratory roles , this seemed like an ideal system to examine such a hypothesis .
Bothfnr and oxrC mutations were found to have distinct effects on expression of different hydrogenase isoenzymes .
Isoenzyme 2 was found to be fnr dependent and oxrC independent , whereas , conversely , isoenzyme 3 was fnr independent , but both activity and formate inducibility were considerably reduced in an oxrC strain .
In addition to regulating the expression of the hydrogenase component of formate hydrogenlyase ( hydrogenase 3 ) , oxrC mutants also have reduced levels of the second enzyme of the system , FDH-BV .
This effect is transcriptional , as shown by the use of an Jhl-lacZ fusion .
These observations are entirely con-a Growth medium consisted of NB and glucose as described in Materials and Methods and cAMP ( 5 mM ) as indicated .
Cells were harvested and immediately subjected to the intact-cell assays as described in Materials and Methods .
b Assays performed on whole-cells .
c Expressed as the specific activity in Triton X-100-dispersed membrane fractions , for the hydrogenase activity that immunoprecipitated by was not antibodies specific for either hydrogenase isoenzyme 1 or 2 ( see Materials and Methods ) .
sistent with the effects of the fnr awnd oxrC mutations on hydrogen metabolism ( respiratory hydrogen uptake is fnr dependent , whereas fermentative hydrogen evolution is oxrC dependent ) and thus provide strong support for a role for isoenzyme 2 in hydrogen uptake and for isoenzyme 3 in hydrogen evolution catalyzed by formate hydrogenlyase .
Hydrogenase isoenzyme 1 is , unusually , dependent on both fnr and oxrC .
Previously ( 24 ) , we proposed that isoenzyme 1 may serve a role in hydrogen recycling .
We envision that hydrogen produced by formate hydrogenlyase activity during fermentative growth is recaptured by isoenzyme 1 and used to reduce endogenously synthesized electron-acceptors .
This isoenzyme therefore serves both fermentative and respiratory roles .
The above results provide good evidence for the pleiotropic nature of the oxrC-dependent anaerobic pathway and its distinction from the fnr-dependent pathway .
In addition , the results provide excellent support for the suggestion ( 7 ) that fnr and oxrC define two distinct anaerobic regulatory pathways with different physiological roles ; fnr-dependent enzymes serve essentially respiratory functions , whereas oxrC-dependent enzymes serve fermentative or biosynthetic roles .
Thus , the fermentative hydrogenase ( isoenzyme 3 ) is oxrC dependent , whereas the respiratory hydrogenase ( isoenzyme 1 ) is fnr dependent .
Significantly , isoenzymne 1 , which serves both a respiratory and a fermentative role , is dependent on both fnr and oxrC .
Why should transcription of anaerobically induced genes whose products serve fermentative roles be under a separate control system to that of genes with respiratory functions ?
The most probable explanation is that , although respiratory proteins are normally only required under very specific conditions ( for example , anaerobically in the presence of the appropriate electron-acceptor ) , the transcription of genes encoding fermentative enzymes is required at a basal level at all times but must be increased to higher levels under specific conditions ( e.g. , anaerobiosis in the absence of an electron-acceptor ) .
In addition , enzymes like formate hydrogenlyase are required at high levels anaerobically only in the absence of respira tion .
Our data suggest that the fermentative enzymes are regulated by fermentative pathways ( glycolysis ) , while it has been suggested that respiratory enzymes are regulated by redox potential ( 19 ) .
This provides a further , and logical , distinction between the two classes of anaerobically induc-ible genes .
Despite the demonstrated distinction in the physiological roles of the oxrC-and fnr-dependent regulatory pathways , both systems interact , albeit indirectly , at the level of formate .
For example , fnr mutations exert some effect onfll activity .
This effect is indirect , as activity is fully restored by the addition of exogenous formate , and is interpreted as being due to a reduction in the synthesis of endogenous formate due to the effect of fnr on pyruvate formatelyase activity .
Similarly , oxrC mutations affect fr activity in two ways ; in addition to the direct effect on anaerobic induction , there is a secondary effect prevented by the addition of exogenous formate .
This is again indirect , being due the to metabolic effects of oxrC mutations , which almost certainly reduce endogenous formate synthesis .
We in no way intend to imply that formate serves as an anaerobic signal-molecule .
Because the various hydrogenase isoenzymes serve either fermentative or respiratory roles , a role for catabolite-repression in the regulation of the cellular isoenzyme content might be anticipated .
In addition to the anaerobic regulation of hydrogenase function , we found that hydrogen metabolism is subject to catabolite-repression in a complex manner .
Consistent with its respiratory role , isoenzyme 2 expression is repressed by glucose in a CRP-dependent manner .
However , even in a crp mutant there is still a significant basal level of isoenzyme 2 expression ; this is in contrast to crp-dependent regulation at the lac promoter but similar to that found for the gal operon ( 26 ) .
Formate hydrogenlyase activity and its associated hydrogenase ( hydrogenase 3 ) are apparently regulated in the opposite hydrogenase manner to 2 , being inhibited by the addition of cAMP .
Again , regulation is CRP dependent .
This finding is , of course , consistent with the fermentative role of formate hydrogenlyase .
This suggests that for this isoenzyme the CRP-cAMP complex may serve , unusually , to decrease gene expression .
However , the situation is unclear , since in contrast to the cya mutant , there is no significant inhibition of formate hydrogenlyase activity by cAMP in a wild-type strain .
We have no explanation at present for this unexpected behavior .
Similarly , the expression of isoenzyme 1 also appears to be reduced by the cAMP-CRP complex .
Our data suggest that the CRP-cAMP complex may serve as an activator of expression of one isoenzyme ( hydrogenase 2 ) and , unusually , as a repressor of another ( hydrogenase 3 ) .
An inhibitory role for the CRP-cAMP complex was suggested previously ( 13 ) .
However , the mechanisms by which this is brought about require further clarification .
Thus , the regulation of hydrogenase isoenzyme content by CRP and cAMP is consistent with the proposed physiological roles of the enzymes .
In addition to regulation by anaerobiosis and by the catabolite-repression system , which presumably modify transcription of the hyd structural genes , the activity of all three hydrogenase isoenzymes can be modified by mutations at four additional loci , hydA and hydB ( 11 , 21 , 29 ) , as well as hydC and hydD ( 30 ) .
Whereas nickel metabolism is implicated in the function of some of these loci ( 29 , 30 ) , the roles of the other pleiotropic hyd loci remain unclear , although they probably act at a posttranscriptional level .
Thus , the regulation of hydrogen metabolism in enterobacteria is extremely complex .
The cellular content of each of the hydrogenases is regulated in a distinct and very precise manner , such that each is synthesized only under growth-conditions in which its activity is required .
Hydrogen metabolism in mutants defective in catabolite-repression Sp act ( U/mg of protein ) of : Formate Nonimmunoprecipitable hydrogenlyase b activityc Addition to Strain growth mediuMa LT2 ( Z ) LT2 ( Z ) None cAMP None cAMP None cAMP 1.0 0.79 0.84 0.49 CH1107 ( cya ) CH1107 ( cya ) CH1108 ( crp ) CH1108 ( crp ) 1.30 0.18 1.73 1.70 0.25 < 0.02 0.54 0.36 We are grateful to P. W. Postma for providing bacterial strains .
This work was supported by grants from the Science and Engineering Research Council to D.H.B. ( GR/C25655 ) and from the Medical Research Council to C.F.H. D.J.J. , R.G.S. , and P.A.R. were supported by studentships from the Science and Engineering Research Council .
C.F.H. is a Lister Institute Research Fellow .
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