165 , No. 3 Oxygen-Regulated Stimulons of Salmonella typhimurium by Mu d ( Ap lac ) Operon Fusions Identified ZARRINTAJ ALIABADI , FELICITY WARREN , SANDRA MYA , AND JOHN W. FOSTER * Department of Microbiology , Marshall University School of Medicine , Huntington , West Virginia 25704 Received 22 August 1985/Accepted 27 November 1985 Using the technique of Mu dl ( Ap lac ) - directed lacZ operon fusions , several oxygen-regulated genetic loci were identified in SalmoneUa typhimurum .
Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified .
Based on control by the oxrA and oxrB regulatory loci , the ani-lacZ-fusions were grouped into three classes : class I loci were regulated by both oxr loci , class H genes were regulated by oxrA only , and class m loci were not affected by either regulatory locus .
Several of the ani-lacZ-fusions required growth in complex medium before they exhibited the inducible phenotype .
While the expression of some of these loci was repressed when organisms were grown in nitrate , others were stimulated by nitrate .
Fusions into the hyd and phs loci were identified among the Isolated ani-lacZ-fusions .
Six oxygen-inducible ( oxi ) operon fusions were also identified .
Two of the oxi loci mapped near oxygen-regulatory loci : oxiC near oxrA and oxiE near oxyR .
However , neither fusion appeared to occur within the regulatory locus .
The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined .
Salmonella typhimurium is capable of growth under either aerobic or anaerobic conditions .
Aerobically , oxygen serves as a terminal electron-acceptor .
Anaerobically , S. typhimurium can utilize alternative respiratory chain acceptors such as nitrate or , if these acceptors are not available , will transfer electrons from formate to H + through the formate hydrogen lyase system ( 3 ) .
In addition , numerous physiologic changes occur during shifts between aerobic and anaerobic-growth including carbon source utilization ( 23 ) , mechanisms of electron flow ( 15 ) , alternative biosynthetic pathways ( 10 , 17 ) , and differences in the maintenance of oxidation-reduction balance ( 12 ) .
Recent two-dimensional gel electrophoretic studies in Escherichia coli by Smith and Neidhardt ( 24 , 25 ) and in our own lab with S. typhimurium ( unpublished observation ) have shown that a number of cellular proteins undergo regulation , either induction or repression , by molecular oxygen .
Utilizing Mu d ( Ap lac ) technology , we fused the lacZ gene to several anaerobically inducible and oxygen-inducible genetic loci in S. typhimurium .
MATERIALS AND METHODS Bacterial strains and cultural conditions .
The bacterial strains used during this study are listed in Table 1 .
Cultural conditions , including minimal E medium and complete LB-medium , were as described previously ( 11 , 21 , 27 ) .
Transductions employing P22 HT 105/1-int phage and the methodology for Mu d phage transposition were also described previously ( 13 ) .
The Mu dl derivative Mu dl-8 was described by Hughes and Roth ( 14 ) .
Cotransduction frequencies were determined by screening a minimum of 100 transductants for an unselected marker .
Ampicillin ( Ap ) was used at a concentration of 30 , ug/ml .
Tetracycline ( Tc ) was added to complex medium at 20 p , g/ml and minimal-medium at 10 , ug/ml .
Where indicated , potassium nitrate and fumaric acid were used at concentrations of 20 mM .
Benzyl viologen medium was used as described by Chippaux et al. ( 7 ) .
Chlorate resistance was tested on LB-medium containing 0.5 % glucose and 50 , umol of potassium chlorate per ml under anaerobic conditions .
Anaerobiosis was achieved either by the use of GasPak anaerobic jars ( BBL Microbiology Systems , Cockeysville , Md. ) , by overlaying culture tubes with paraffin oil or by bubbling N2 into culture flasks .
Aerobic conditions were attained by vigorous shaking of 5 ml in a 50-ml flask .
MacConkey 's nitrate medium was used to screen for nitrate reductase , formate dehydrogenase , and glycerol metabolism mutants ( 1 ) .
Incubations were routinely carried out at 30 °C .
Construction of operon fusions .
The basic procedure for isolating oxygen-regulated operon fusions was as follows : P22 phage propagated on Mu dl lysogen ( JF296 ) or a Mu dl-8 lysogen ( SF157 ) was used to transduce JF235 and JF706 , respectively , to Apr on LB plates containing 30 , ug of Ap per ml .
This was done under aerobic conditions at 30 °C .
Subsequently , Apr colonies were replicated to MacConkey lactose medium containing Ap , and duplicate plates were incubated aerobically and anaerobically ( GasPak System ; BBL ) .
Colonies that appeared to produce more 1-galactosidase under anaerobic conditions were purified and designated anaerobically inducible ( ani ) , while strains that produced more,3-galactosidase aerobically were designated oxygen inducible ( oxi ) .
In vitro 1-galactosidase assays were performed as described by Miller ( 21 ) .
All cultures were grown to equivalent optical densities before the assays were performed .
Values represent the average of two independent assays .
Unless otherwise noted , cells were grown in minimal E medium at 30 °C ( 27 ) .
Steady-state conditions were ensured following shifts in oxygenation by allowing at least two or three generations of growth prior to assay .
Most of the 1-galactosidase activities reported were obtained from cultures overlayed with paraffin oil .
Comparisons between activities obtained by using alternate methods to achieve anaerobiosis revealed no significant differences .
Determination of transcriptional orientation .
The technique described by Maloy and Roth ( 20 ) was used to determine the direction of transcription for several of the anaerobically inducible genetic loci .
Hfr strains were constructed by integrating an F'ts lac + TnJO plasmid ( FF21 ) into the ani genes by using sequence homology between the lac operon on the plasmid and lac sequences present in the Mu dl-8 insert .
The orientation of the Mu dl-8 insert dictates the orientation of F-factor integration and , thus , the direction of transfer of the resulting Hfr .
To ensure stability , the ani : : Mu dl-8 fusions were transferred to SF11 before the F'ts lac + TnWO plasmid was introduced .
Recombinants were selected as described by Chumley et al. but with selection for Tcr at 420C ( 9 ) .
Strains used in this study Strain ( source Genotype Construction of oxygen-regulated operon fusions .
Approximately 14,000 Apr transductants were screened for oxygen regulation by the methodology described above .
Thirteen strains proved , on further in-vitro , B-galactosidase measurements , to contain fusions in genes which became induced ( or derepressed ) during anaerobic-growth , while six strains appeared to contain oxygen-inducible fusions ( Table 2 ) .
The inducible levels of the ani-lacZ-fusions varied from 2-to 150-fold over repressed levels , depending on the strain examined .
The oxygen-inducible fusions , however , only responded between two-to sixfold to anaerobic-aerobic shifts .
Figure 1 presents results typically obtained during kinetic studies .
Strains containing ani-lacZ-fusions responded almost immediately to shifts to anaerobiosis , while the reverse was found for oxi-lacZ strains .
The ani-lacZ operon fusions were examined for their response to nitrate , fumarate , and chlorate .
Many known oxygen-regulated genes are repressed by nitrate under anaerobic conditions , and mutations in several oxygenregulated genes involved with nitrate reductase result in chlorate resistance under anaerobic conditions ( 2 , 4 , 6 , 16 , 30 ) .
Table 3 presents the results of these studies .
Most anilacZ-fusions were repressed to various degrees by nitrate .
Fusions ani-2 : : Mu dl-8 , ani-1071 : : Mu dl-8 , aniG1072 : : Mu dl-8 , and aniE1074 : : Mu dl-8 were not significantly repressed by nitrate .
The fusions in strains JF900 , JF1103 , and JF1106 , however , were unusual in that the presence of nitrate enhanced induction under anaerobic conditions .
These fusions were also unusual in that induction could only be demonstrated in complex LB-medium .
It is unlikely that these fusions occur in genes involved with nitrate reductase ( chl ) which require nitrate as well as anaerobiosis for their induction .
The ani-lacZ operon fusions in question were not resistant to chlorate and were not induced by nitrate in minimal-medium .
In addition , they appeared normal on the MacConkey nitrate agar described by Barrett et al. ( 1 ) .
The phenomenon was not related to glucose repression , as initially suspected , because the addition of 1 % vitamin-free Casamino Acids ( CAA ; Difco Laboratories , Detroit , Mich. ) to minimal salts glucose allowed for anaerobic induction ( Table 4 ) .
Nitrate also accentuated induction in CAA-supplemented ruedium with JF900 but had no effect in this medium with JF1103 or JF1106 .
Two other ani-lacZ operon fusions ( JF1105 and JF1107 ) were found to require growth in LB-medium to exhibit anaerobe inducibility ( Table 4 ) .
Anaerobic induction of strain JF1105 ( aniGJ072 ) was found to be repressed by glucose when it was added to LB-medium ( data not shown ) .
The fusion in JF1107 ( aniE ) required either CAA or LB-medium for anaerobic induction , but glucose partially repressed induction in LB-medium .
Results of a study by Lester and DeMoss ( 19 ) suggest that the induction of nitrate reductase , as well as components of the formate hydrogenlyase system under anaerobic conditions , are dependent on the presence of selenite , molybdate , or both .
This does not appear to be the case with the fusions that require complex medium for induction under anaerobiosis .
Neither sodium selenite ammonium nor molybdate , added to 10-6 M to minimal-medium , had any effect on the induction of these strains .
Fumarate did not appear to significantly affect the expression of most of the ani-lac operon fusions described here ( Table 3 ) .
However , the fusion in JF1104 responded to fumarate with an approximate twofold enhancement of anaerobic induction .
Map positions and direction of transcription of oxygenregulated genes .
Strains containing TnJO insertions near various ani and oxi loci were isolated by crossing each fusion strain with P22 phage propagated on a pool of random TnJO insertions and selecting for Lac-Aps transductants .
These insertions were transferred to various Hfr strains via transduction , and the resultant strain was used in interrupted mating experiments designed to transfer both Tcr and known auxotrophic markers .
Alternatively , phage preparations grown on 60 strains containing known TnJO or TnS insertions were used to map some of the fusions .
In addition , Mu dl-8 insertions were utilized in some strains to determine the direction of transcription of some of the ani loci by constructing lac-directed Hfr strains , as described by Maloy and Roth ( 20 ) .
The results of these experiments are presented in Fig. 2 .
Two of the ani-lacZ-fusions ( aniA3 and aniA1055 ) were found to map at or near the hyd locus of S. typhimurium based on their cotransduction with srl-202 : : TnJO ( 43 % ) .
Characteristically , for hyd mutants , these strain-s did not reduce benzyl viologen under anaerobic conditions ( 30 ) .
However , recent work with E. coli has revealed an additional locus near hyd , designated ant , which is involved with anaerobic electron transport ( 30 ) .
A-Galactosidase activities of anaerobically and aerobically inducible gene fusions-Galactosidase activity ' Fold dere-Aerobic Anaerobic pr.ession JF556 ani-2 : : Mu dl-8 280 509 2 JF738 aniA ( hyd ) -3 : : Mu dl 12 419 35 JF895 aniD1047 : : Mu dl-8 6 334 56 JF9000 aniC1052 : : Mu dl-8 29 226 7.7 JF926 aniB1054 : : Mu dl-8 15 538 34 JF927 aniA ( hyd ) lO55 : : Mu dl-8 12 1163 92 JF11O1 aniF1068 : : Mu dl-8 44 672 15 JF1102 aniH1069 : : Mu dl-8 21 136 6 JF1103a aniJlO70 : : Mu dl-8 1 131 131 JF1104 ani-1071 : : Mu dl-8 21 226 11 JF1105a aniG1072 : : Mu dl-8 198 1250 63 JF11060 ani-1073 : : Mu dl-8 1 71 71 JF1107 ' aniE ( phs ) 1074 : : Mu dl-8 21 164 8 JF739 oxiE4 : : Mu dl 379 192 2 JF888 oxi-1046 : : Mu dl-8 518 235 2 JF896 oxiC1048 : : Mu dl-8 715 281 2.5 JF897 oxiA1049 : : Mu dl-8 1,080 224 5 JF9014 oxiD1053 : : Mu dl-8 792 199 4 JF928 oxiBIOS6 : : Mu dl-8 689 110 6 Regulation was only observed in complex media ( LB ) .
b Activity is in Miller units Strain Fusion ALIABADI ET AL. .
c E 0a 0 0 E - U CM C 0 0 to Q ) .0 3 .7 Q 0 o 0 U ) 0i u 0 0 Ct 0 0 2 8 8 4 6 2 4 6 Time ( Hours ) Time ( Hours ) o FIG. 1 .
Kinetics of ani-lacZ and oxi-lacZ induction .
( A ) Strain JF738 ( ani-lacZ ) was grown in minimal-medium with vigorous aeration .
The arrow indicates the point at which the culture was split .
Nitrogen gas was bubbled through half of the culture to achieve anaerobiosis .
( B ) Strain JF897 ( oxiA-IacZ ) was initially grown overnight aerobically , and then at time zero fresh minimal-medium was inoculated and grown anaerobically ( bubbled nitrogen gas ) .
At the time indicated with an arrow , half of the culture was aerated vigorously .
Circles represent growth , squares represent P-galactosidase activities , open symbols represent anaerobic conditions , and closed symbols represent aerobic conditions .
ODw , Optical density at 660 nm .
BL TA Effect of nitrate and fumarate on ani-lac expression P-Galactosidase activity Anaerobica Strain Genotype Aerobic No With nitrate With fumarate nutrients JF556 ani2 : : Mu dl-8 226-689-772 JF738 aniA3 : : Mu dl 12 465 49 JF895 aniD1047 : : Mu dl-8 8 274 163 JF9OOb aniC1052 : : Mu dl-8 21-259-756 JF926 aniB1054 : : Mu dl-8 11-338-118 JF927 aniA1055 : : Mu dl-8 21-872-110 JF1101 aniF1068 : : Mu dl-8 31-455-292 JF1102 aniH1069 : : Mu dl-8 43 172 51 JF1103b aniI1070 : : Mu dl-8 4 124 232 JF1104 anilO71 : : Mu dl-8 18 94 130 JF1105b aniG1072 : : Mu dl-8 215-790-735 JF1106b anilO73 : : Mu dl-8 1 88 213 JF1107b aniEJ074 : : Mu dl-8 23-145-115 a Nitrate was added to 20 mM ; fumarate was added to 20 mM .
b In complex media ( LB ) 661-515-282-176 461-928-579-196 84-173-691 81 202 FIG ; 2 .
Linkage map of oxygen regulated loci in S. typhimurium .
The relationship of loci marked with an asterisk to adjacent markers is not known .
Map locations of loci in parentheses are approximate .
We do not know whether the insertions isolated occur in an analogous S. typhimurium ant locus .
Using lac-directed Hfr formation as outlined by Maloy and Roth ( 20 ) , the direction of transcription for aniA was determined to be counterclockwise ( Fig. 2 ) .
The aniB-lacZ fusion ( JF926 ) was mapped at 93 units by cotransduction to zid-27 : : TnlO ( 15 % ) and was found to be transcribed counterclockwise .
The fusion in JF900 ( aniC ) was 98 % linked to zid-27 : : TnJO at 93 units and also had a counterclockwise transcriptional orientation , while aniD ( JF895 ) mapped between 90 and 95 units ( unlinked to zid-27 : : TnJO ) , with transcription occurring in a clockwise direction .
Strauch et al. ( 26 ) have recently reported the isolation of several anaerobically inducible operon fusions in S. typhimurium which they refer to as oxd .
Their selection procedure differed from ours , as did the map positions , the phenotypic properties , or both of several of their loci when compared with our ani genes .
Two genes with similar map positions to an oxd locus are aniB and aniC which are located at 93 units near oxdB .
In contrast to oxdB , aniC required CAA and nitrate for full derepression under anaerobic conditions .
Thus , we believe that these two loci are distinct and separate .
The aniB locus , however , could be analogous to oxdB .
Similarly , Barrett et al. ( 2 ) have described two anaerobically inducible gene fusions in S. typhimurium .
One fusion , they suggest , occurred in the hyd locus , although they did not address the possibility of an ant fusion which would produce the same phenotype .
They also isolated a strain containing an operon fusion in a flu locus located at 93 units .
Full expression of this fusion required both formate and anaerobiosis , while the addition of nitrate repressed transcription of this gene .
It is possible that our aniB gene and the oxdB locus of Strauch et al. ( 26 ) could be analogous to this f7hl locus .
H4owever , the aniC locus we describe must be a separate locus because nitrate serves to enhance its expression in th-e presence of CAA .
In addition , whereas an Ihl insertion prevents the production of gas in triple sugar-iron agar , the aniB fusion , when placed in an LT2 background , still allows the production of gas , indicating that aniB is probably distinct from Jhl .
The aniE ( phs ) - lacZ fusion was mapped at 41 units based on an 85 % cotransduction with zec-2 : .
The aniE locus appears to be similar to the phs locus reported by Voll et al. ( 28 ) which is involved with the production of hydrogen sulfide .
Transduction of the aniE : : Mu dl-8 fusion to LT2 resulted in 100 % cotransduction of the H2S negative phenotype , as tested in triple sugar-iron agar .
These studies indicate that the transcription of the phs locus is regulated under anaerobic conditions but not by the oxrA or oxrB gene products ( see below ) .
It is also of interest to note that thi locus is only induced anaerobically in complex media ( Table 4 ) .
Two additional fusions were found to map near metC ( aniF and aniG ) at 63 units .
Cotransduction frequencies of aniF and aniG to a metC : : TnJO insertion ( SF240 ) were 20 and 3.5 % , respectively .
One or both of these Mu d insertions might occur in the oxdA locus described by Strauch et al. ( 26 ) .
However , in contrast to oxdA , aniF requires complex media for induction , and neither aniF nor aniG appears to be regulated by oxrA or oxrB .
Thus , it would seem that aniF and aniG represent loci distinct from oxdA .
The direction of transcription for aniG was determined as being clockwise based on the construction of lac-directed Hfr strains ( Fig. 2 ) .
Several of the oxygen-inducible loci were also mapped .
The oxiA locus was mapped between 9 and 13 units , oxiB at 22 units , and oxiC at 30 units .
Cotransduction studies revealed that oxiB was 90 % linked to putA : : TnlO .
Maloy and Roth ( 20 ) have determined that the oxygen regulation of the proline utilization genes is by the putA gene product .
The oxiB strain does exhibit a Put phenotype , and its induction is controlled by putA because a putA : : TnJO insertion severely reduces the expression of oxiB .
However , the oxiB fusion was not subject to catabolite-repression and was not dependent on proline for induction , as observed for putA or putC .
The oxiC locus was cotransducible ( 98 % ) with a TnWO insertion near the anaerobic regulatory oxrA locus described below ( SF165 ) and therefore maps at 30 units .
The oxiE locus mapped at 88 units and was cotransducible with argH : : TnJO ( 65 % ) .
This places oxiE near the oxyR regulatory locus described below .
Although Barreott et al. ( 2 ) described a pyrA : : Mu dl operon fusion which appeared to be oxygen inducible , none of our oxi-lacZ-fusions were cotransducible with pyrA : : TnlO .
Effect of various regulatory mutations on ani-lacZ and oxi-lacZ expression .
Studies with E. coli have implicated that a locus designated nirR ( or fnr ) is in control of several oxygen-regulated genes ( 18 ) .
The nirR gene product appears to be a positive-acting regulatory factor that is essential for the expression of several anaerobic enzyme systems ( 6 ) .
A similar mutation in a gene designated oxrA has been identified by Strauch et al. ( 26 ) in S. typhimurium .
The oxrA gene product appears to be a positive regulator of the anaerobe-inducible ( oxd ) fusions that were isolated by Strauch et al. ( 26 ) .
Consequently , we wanted to determine the effect of the oxrAI mutation on the ani-lacZ-fusions identified in this report by transducing oxrA from SF165 into our strains .
The results suggest that although the oxrA mutations reduce the-amount of derepression obtained with many of the ani-lacZ-fusions ( ani-2 , ani-1068 , aniH , ani-1072 , and aniE were not affected by oxrA ) , most of the fusions affected by oxrA still exhibit considerable derepression , suggesting additional regulation of these loci ( Table 5 ) .
Strauch et al. ( 26 ) have described a secondary regulatory locus designated oxrB which also prevented the induction of oxygen-regulated loci .
Using a TnS insertion near oxrB8 ( SF233 ) , we transferred oxrB8 to our operon fusions .
The only ani-lacZ strains that were affected are listed in Table 5 along with their , B-galactosidase activities .
Of 13 anaerobe-inducible fusions , only 4 were clearly affected by oxrB .
This is in contrast to the results of Strauch et al. ( 26 ) in which all of their oxd fusions required oxrB for anaerobic induction .
Buck and Ames ( 5 ) have identified a locus ( miaA ) which codes for an isopentenylation enzyme involved with the modification of the adenosine residue ( position 37 ) adjacent to the anticodon in tRNAs which recognize codons beginning with U .
The hydroxylation of this group varies , depending on the oxygen state of S. typhimurium .
In aerobically grown cells , the tRNAs contained cis-2-methylthioribosyl-zeatin ( ms2io6A ) .
Anaerobically , the hydroxylation of the isopentenyl side chain does not occur ( ms2i6A ) .
These authors hypothesize that the fluctuations in tRNA modification could influence attentuation control of transcription .
Consequently , we transferred several of our oxi-lacZ operon fusions to a miaA strain provided by M. Buck .
None of the fusions tested ( oxiA , oxiC , oxiD ) , however , appeared to be significantly affected by the miaA mutation .
In general , the expressions of these fusions were reduced in the mia deriv-ative strains , but the relative inductions were comparable with those of the miaA + counterparts .
Another regulatory locus ( oxyR ) identified by Christman et al. ( 8 ) was shown to be involved in the positive control of an oxidative-stress defense regulon as well as a subset of heat-shock proteins in S. typhimurium .
The possibility that some of the oxi-lacZ-fusions may be controlled by oxyR was tested by introducing oxyRI into the fusion strains .
While measurements of P-galactosidase activities failed to show any regulation by oxyRI , one of the fusions ( oxiE ) was found to map close to oxyRI as described above .
The oxiE locus does not appear to be analogous to oxyR because the oxiE : : Mu dl-8 insertion did not result in an increased sensitivity to hydrogen-peroxide ( 8 ) .
Effect of nitrate and CAA on 3-galactosidase activities during ani-lacZ induction Effect of 02 on the following media with the indicated additions : LB Minimal O Strain None NO3a CAA NO3 + CAA +02 -02 6 938 1 244 25 17 1 298 1 11 N 3 None +02 -02 +02 -02 +02 -02 JF900 < 1 < 1 < 1 < 1 4 371 JF1103 5 3 5 1 1 262 JF1105 17 19 25 18 25 52 JF1106 < 1 < 1 < 1 < 1 1 247 JF1107 2 3 4 1 1 64 a Nitrate was added at 20 mM ; CAA ( vitamin-free CAA ) was added at 1 % .
+02 44 1 204 1 23 -02 874-343-735-314 116 +02 39 1 215 1 18 -02 350-157-790-183 144 1-Galactosidase activitya Strain Genotype Media +02 -0 ol 2 induction 20 645 32 10 145 15 JF738 aniA3 : : Mu dl Minimal JF1053 aniA3 : : Mu dl oxrAI Minimal JF895 aniD1047 : : Mu dl-8 Minimal 8 93 12 JF1054 aniD1047 : : Mu dl-8 oxrAI Minimal 6 12 2 JF1114 aniD1047 : : Mu dl-8 oxrB8 Minimal 5 3 1 JF900 aniC1052 : : Mu dl-8 JF1055 aniC052 : : Mu dl-8 oxrAI JF1115 aniC1052 : : Mu dl-8 oxrB8 JF926 aniBI054 : : Mu dl-8 JF1056 aniB1054 : : Mu dl-8 oxrAI JF1117 aniB1054 : : Mu dl-8 oxrB8 LB + NO3 LB + NO3 LB + NO3 Minimal Minimal Minimal 4 5 9 228 60 11 63 13 1 11 8 4 316 38 25 29 5 6 JF927 aniA1055 : : Mu dl-8 Minimal 7 549 80 JF1065 aniA1055 : : Mu dl-8 oxrAI Minimal 6 145 26 JF1116 aniAlOSS : : Mu dl-8 oxrB8 Minimal 4 372 83 JF1103 aniI1070 : : Mu dl-8 JF1112 aniI1070 : : Mu dl-8 oxrAI JF1118 aniJlO70 : : Mu dl-8 oxrB8 a Activity is in Miller units ( 21 ) .
LB 2 125 73 LB 2 59 27 LB 5 10 21 DISCUSSION The results presented in this report and those of others ( 16 , 26 , 30 ) indicate that there is a complex set of controls which regulate the anaerobic stimulon of S. typhimurium .
Relative to the positive regulators oxrA and oxrB there are three classes of anaerobe-inducible genes ; class I loci are regulated by both oxrA and oxrB , ( e.g , aniD ) , class II-loci are regulated by oxrA only ( e.g. , aniA ) , and class III loci are not dependent on either regulatory protein ( e.g. , aniE [ phs ] o aniF ) , indicating additional control mechanisms .
We also uncovered a unique group of anaerobically inducible loci which require the presence of CAA ( or growth in LB-medium ) for induction ; some of these loci have induction ratios ( anaerobic p-galactosidase activity/aerobic Pgalactosidase activity ) that are enhanced by the presence of nitrate .
One possibility is that these loci represent oxygenregulated peptidases or peptide-permeases similar to tppB described by Jamieson and Higgins ( 16 ) and pepT described by Strauch et al. ( 26 ) .
However , pepT-lacZ induction does not require CAA , and tppB-lacZ induction is repressed , not enhanced , by the addition of nitrate .
It should be noted , however , that tppB represents another anaerobically controlled locus that is not regulated by oxrA .
Very little information is available concerning the genetics of H2S production in S. typhimurium .
The proteolysis of proteins and peptides such as those found in CAA results in the production of cysteine , which can be catabolized via cysteine desulfurase to pyruvic acid , hydrogen sulfide gas , and ammonia .
Alternatively , thiosulfate can be reduced via anaerobic respiration , whereby the sulfur atom serves as the electron-acceptor for the oxidation of organic substrates yielding sulfite and hydrogen sulfide gas ( 22 ) .
There have been no studies dealing with the regulation of H2S production .
The studies presented here with the aniE ( phs ) - lacZ of by S. fusion strain indicates that the production H2S typhimurium is controlled at the transcriptional level and requires the presence of organic medium and anaerobic conditions for full expression .
T-his is also the first report of loci that have been identified initially on the basis of their oxygen inducibitity .
Six loci were identified , four of which were mapped .
The oxiB fusion maps near putA , a locus known to have its transcription enhanced by growth on oxygen .
Two of the oxi-lacZ-fusions oxygen-regulatory loci .
oxiC mapped near oxrA , map near which regulates several anaerobically inducible genes , while oxiE mapped near oxyR , which controls the cellular response to-oxidative-stress .
This may just be coincidence , but studies are under way to determine whether there is a more direct functional relationship between these loci .
The discovery by Buck and Ames ( 5 ) of a tRNA modification occurring during shifts from anaerobic to aerobic conditions suggests the possibility of an attenuation-like mechanism that controls the expression of some oxygenregulated genetic loci .
Our results with oxiA , oxiC , and oxiD-indicate that this tRNA modification probably does not contribute significantly to the oxygen regulation of these genes .
A recent report by Yamamoto and Droffner ( 29 ) indicates that supercoiling of the bacterial chromosome also plays an important role in promoter selection under aerobic and anaerobic conditions .
Mutants of S. typhimurium isolated as found to contain mutations in gyrA or strict aerobes were gyrB ( DNA gyrase ) while mutants isolated as strict anaerobes contained mutations in topA ( topoisomerase I ) .
Thus , somne of the transcriptional responses observed with ani-lacZ and oxi-lacZ-fusions may reflect alterations that occur in chromosomal supercoiling rather than regulation by diffusible regulatory proteins .
The procedures outlined above and those described by Strauch et al. ( 26 ) have enabled the characterization of oxygen-regulated genetic loci without the need to numerous identify a oxygen-related specific phenotype .
Use of these techniques will ultimately lead to a more detailed picture of the aerobic and anaerobic stimulons of S. typhimurium and the regulatory circuits which control their expression .
JF927 aniA1055 : : Mu dl-8 Minimal 7 549 80 JF1065 aniA1055 : : Mu dl-8 oxrAI Minimal 6 145 26 JF1116 aniAlOSS : : Mu dl-8 oxrB8 Minimal 4 372 83 JF1103 aniI1070 : : Mu dl-8 JF1112 aniI1070 : : Mu dl-8 oxrAI JF1118 aniJlO70 : : Mu dl-8 oxrB8 a Activity is in Miller units ( 21 ) .
LB 2 125 73 LB 2 59 27 LB 5 10 21 We thank J. Gots , J. Roth , C. Miller , B. Ames , and K. Hughes for kindly providing strains for our use in this study .
This work was supported by Public HIealth Service grant GM 34147-01 from the National Institutes of Health .
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