The poc Locus Is Required for 1,2-Propanediol-Depende The enteric bacterium Salmonella typhimurium can synthesize cobalamin ( vitamin-B12 ) de novo , but only under anaerobic-growth-conditions ( 14 ) .
The main cluster of coba-lamin biosynthetic ( cob ) genes ( at 41 min ) has been divided into three regions , CobI , CobII , and CobIII .
The cobI genes are required for the synthesis of adenosyl-cobinamide ( Ado-Cbi ) ; the cobII genes are required for the synthesis of the nucleotide dimethylbenzimidazole ; and the cobIII genes are required to join Ado-Cbi and dimethylbenzimidazole to form adenosyl-cobalamin ( 10 , 14 ) .
Previous work has shown that the Cob regions are contiguous in the order CobI , CobIII , and Cobll ; that they are transcribed counterclockwise ; and that internal promoters allow basal , constitutive expression of cobII and cobIII genes under aerobic-growth-conditions ( 8 , 14 ) .
S. typhimurium does not make Ado-Cbi ( the proposed end product of the CobI pathway ) de novo in the presence of oxygen ( 14 ) .
Unlike CobI functions , CobII and CobIII functions are available for the synthesis of adenosyl-coba-lamin under aerobic-growth-conditions provided that cobin-amide is supplied in the culture medium .
The reasons for the lack of synthesis of Ado-Cbi during aerobic-growth of this bacterium have been investigated to a limited extent .
The analysis of cobI transcription by using lacZ-transcriptional-fusions to the CobI promoter indicated that expression of the cobI genes was conditional to the absence-of-oxygen in the environment ( 9 ) .
It appeared from these results that the inability to synthesize Ado-Cbi under aerobic conditions was due to lack of transcription of the CobI region .
However , Andersson and Roth ( 1 ) reported the isolation of mutants capable of transcribing the cobI genes to levels-280-fold higher than in the wild-type strain .
These mutants were still unable to synthesize Ado-Cbi under aerobic-growth-conditions , which suggested that increasing the transcription of cobI was not sufficient to allow Ado-Cbi synthesis under aerobic conditions .
One metabolic pathway of S. typhimurium which requires cobalamin is the catabolism of 1,2-propanediol ( 1,2-PDL ) .
1,2-PDL can serve as the sole carbon and energy source for the cell under aerobic-growth-conditions , provided that the cells are supplemented with cobinamide or cobalamin .
In this bacterium , the catabolism of 1,2-PDL proceeds via the cobalamin-dependent propanediol dehydratase , which converts it to propionaldehyde , which is then oxidized to propionate ( 13 ) .
The genes required for the catabolism of 1,2-PDL to propionate ( PDL utilization or pdu genes ) are located at 41 min , upstream of the cob operon .
The physical distance between pdu and cob is unknown , but genetic markers in the two regions have been reported as being 12 % cotransducible by bacteriophage P22 ( 13 ) .
It is also known that the pdu and cob genes are divergently transcribed and that pdu expression is regulated at the transcriptional level by 1,2-PDL ( 13 ) .
In this paper , we present evidence that 1,2-PDL is a positive effector of cob transcription and report the isolation and initial characterization of mutants unresponsive to the stimulatory effects of 1,2-PDL .
These mutants carry lesions that define a new locus referred to as poc ( PDL and cobalamin ) .
We propose that expression of the cob and pdu genes is coregulated at the transcriptional level by the poc gene product ( s ) in response to 1,2-PDL .
( Part of this work was presented at the 1991 American Society for Microbiology General Meeting in Dallas , Tex. [ 16a ] .
) MATERIALS AND METHODS Bacterial strains , media , and growth-conditions .
All bacterial strains used in this study are derivatives of S. typhimu-rium LT2 and are listed in Table 1 .
As detailed in Table 1 , all cob-24-lac strains are derivatives of JE1734 and all pdu-8-lac strains are derivatives of RT818 .
Nutrient broth ( 0.8 % [ wt/vol ] ; Difco , Detroit , Mich. ) containing 85 mM NaCl was used as complex medium .
No-carbon E medium supplemented with 1 mM MgSO4 was used as the minimal-medium JE1734 JE1929 poc-102 : : TnlOd ( Tc ) JE1933 poc-106 : : TnJOd ( Tc ) JE1947 recAl derivative of JE1929 JE1951 recAI derivative of JE1933 JE1952 recAl JE1958 JE1947/F ' poc + JE1962 JE1951/F ' poc + JE1963 JE1952/F ' poc + a Unless otherwise stated , the strains listed were obtained during the course of this study .
b TnlOd ( Tc ) , TnlODEL16DEL17 ( Tc ) .
c The F ' plasmid strain JE1968 is refered to as F ' poc + in all other strains .
Unless otherwise stated , when provided in the medium , the final concentrations of compounds in the culture medium were as follows : glycerol , 22 mM ; succinate , 35 mM ; ace-tate , 10 rpM ; glucose , 11 mM ; pyruvate , 50 mM ; sodium nitrate , 5 & mM ; 1,2-PDL , 12 mM ; 1,2-PDL analogs , 6 mM ; methionine , 0.5 mM ; 5-bromo-4-chloro-3-indolyl-13-D-galac-topyranoside ( X-Gal ; Bachem , Inc. , Torrance , Calif. ) 20 mg/liter ; and lactose , 2.5 mM .
In some cases ( indicated below ) acetate was added to the medium in addition to the main carbon and energy source to increase the rate of growth .
Antibiotics in rich-medium ( per ml ) were as follows : kanamycin , 50 , ug ; ampicillin , 30 , ug ; tetracycline , 20 , ug ; and chloramphenicol , 20 , ug .
In minimal-medium , tetracycline was used at a concentration of 10 , g/ml .
For cultures grown anaerobically on glucose , the glucose concentration was 22 mM .
Regardless of the carbon and energy source used , all anoxic media also contained NaHCO3 ( 40 mM ) and trace minerals solution ( 10 ml/liter of medium ) ( 3 ) .
Cell growth of anaerobic cultures was monitored with a Spectronic 20D spectrophotometer ( Milton Roy Co. , Rochester , N.Y. ) , with a light path of 1.5 cm .
Cultures at anA650 of between 0.15 and 0.20 were in early log phase .
Aerobic cultures were routinely grown in 5 ml of medium in a 125-ml Klett flask and vigorously shaken ( 400 rpm ) to ensure high aeration .
Cell growth for aerobic cultures was monitored with a Klett-Summerson colorimeter equipped with a red filter .
An early-log-phase culture corresponded to a reading of 55 to 70 Klett units .
The typical inoculum size used for growing cells to measure transcription of the cob and pdu genes was about 2 % ( vol/vol ; ca. 108 cells ) .
Unless otherwise stated , inocula were started from isolated single colonies grown overnight in the same medium to be used for outgrowth .
Growth with 1,2-PDL as the sole carbon and energy source was determined by using NCE minimal-medium containing 1,2-PDL ( 53 mM ) , CN-B12 ( 15 nM ) , and MgSO4 and methionine as described above .
Cultures were routinely grown at 30 °C .
A lysate of the high-transducing mutant bacteriophage P22 HT 105/1 int-201 ( 18 , 19 ) was as prepared described elsewhere ( 7 ) on a pool of approximately 50,000 strains , each carrying one insertion of the transpositiondefective element TnlODEL16DEL17 ( Tc ) ( 21 ) [ hereafter referred to as TnlOd ( Tc ) ] .
This lysate was used as donor to transduce JE1734 to tetracycline resistance .
Transduction plates were replica printed onto minimal-medium plates containing lactose with or without 1,2-PDL ; alternatively , Tcr colonies were directly selected on minimal-medium with succinate , X-Gal , tetracycline , and 1,2-PDL .
Methionine was added to satisfy the auxotrophic requirement of the strains used .
Putative mutants were freed of contaminating phage on green indicator plates ( 7 ) .
Equal volumes of donor and recipient full-density cultures ( 0.1 ml , ca. 108 cells ) were mixed on a nutrient broth plate , incubated for 4 h at 37 °C , and replica printed onto nutrient broth plates containing the appropriate antibiotic .
Chloramphenicol was used to select for transfer of the F ' plasmid , and either kanamycin or ampicillin was used to counterselect against the donor .
Exconjugants were purified nonselectively on nutrient broth plates , and their phenotypes were tested .
13-Galactosidase enzyme activity assays .
, B-Galactosidase enzyme activity was determined by the method of Miller ( 15 ) in CHCl3-sodium dodecyl sulfate-permeabilized cells as described elsewhere ( 9 ) .
Routinely , cells were pelleted by centrifugation at 4,000 x g for 10 min in a bench-top low-speed centrifuge ( Sorvall ; model GLC-2 ) and kept on ice until assayed .
Cells were resuspended in 2 ml of sterile saline for the assay .
Units of 0-galactosidase activity are reported per A60 unit of the culture .
RESULTS The experimental evidence presented in this communication is consistent with the existence of a 1,2-PDL-dependent regulatory system for the transcriptional control of both the cob and pdu genes .
A highlight of this regulatory system is that it allows high levels of transcription of the cobI ( Ado-Cbi biosynthesis ) genes in the presence of oxygen .
1,2-PDL-dependent transcription of the cob genes .
( i ) Effect of 1,2-PDL on cob transcription .
Under highly aerated growth-conditions , the presence of 1,2-PDL in the culture medium resulted in a significant increase in the transcription of fusions to all three Cob regions ( Table 2 ) .
CobI transcription increased dramatically ( 38-fold ) when 1,2-PDL was present in the medium .
CobIl and CobIII transcription increased eight-and ninefold , respectively , in response to 1,2-PDL in the medium .
This pattern of regulation , in which effectors of cobI transcription have a more pronounced effect on Cob a The basal medium used for all cultures was NCE-succinate containing methionine .
Conditions for the preparation of the inoculum and its outgrowth are described under Materials and Methods .
3-Galactosidase activity was assayed in early-log phase cultures .
than over CobIl or CobIIL , is consistent with previously reported studies on the transcriptional regulation of the cob genes ( 9 ) .
All of the experiments were performed with cultures grown at 30 °C in light of our observations which indicated that an inverse relationship existed between the stimulatory effect of 1,2-PDL and temperature ( data not shown ) .
Under anoxic conditions , the presence of 1,2-PDL in the medium caused a fourfold induction in the transcription of the cobI-lac fusion and sevenfold induction in the transcription of the cobII-lac and cobIII-lac fusions ( data not shown ) .
Expression of the cobI-lac fusion in the presence of 1,2-PDL was similar under aerobic and anaerobic conditions .
The lower value for induction of the cobI-lac fusion ( 38-fold aerobically versus 4-fold anaerobically ) was the result of a higher level of expression of the fusion under anaerobic conditions without added 1,2-PDL ( 110 U anaerobically versus 15 U aerobically ) .
These data strongly suggest that 1,2-PDL is a positive regulator of transcription of all three regions of the cob operon under both aerobic and anaerobic-growth-conditions .
( ii ) Effect of 1,2-PDL on other genes .
1,2-PDL had no stimulatory effect on the expression of operon fusions to cobA , a gene involved in cobalamin biosynthesis located outside the cob operon ( 10 ) , or to btuB , a gene required for cobalamin transport across the outer membrane ( 4 , 12 ) ( data not shown ) .
The only other genes whose transcription has been found to be regulated by 1,2-PDL were the PDL utilization ( pdu ) genes ( 13 ) .
The specificity of the response to 1,2-PDL was examined by measuring the level of transcription of fusion cob-24-lac ( CobI ) in cultures grown aerobically in the presence of compounds structurally related to 1,2-PDL .
We tested the following compounds : propan-1-ol , 1,3-PDL , ethylene glycol , glycerol , propionate , propionaldehyde , and ethanolamine .
The average level of 3-galactosi-dase activity measured in cultures grown in medium containing any one of these compounds was 14 + 2 U per A650 U .
This average was the same as the level measured in the absence of any additions ( 15 U perA650 U ) .
It is possible that in some cases the failure to induce cobI transcription reflected a lack of transport of the compound in question .
Even though this possibility was not addressed , it is known that S. typhimurium can grow on ( and therefore transport ) ethanol-amine , propionate , and glycerol ( 11 , 17 ) .
We also compared the effects of R ( - ) - and S ( + ) -1,2-PDL on cobI transcription ( Table 3 ) .
We measured a 30-fold increase in the transcription of fusion cob-24-lac when S ( + ) -1,2-PDL was present in the culture medium , a 18-fold increase in transcription of the fusion was measured when 3-Galactosidase activity Of JE1734 Induction Of RT818 Induction Growth6 ( U/A650 U ) ( fold ) ( U/A650 U ) ( fold ) None 20 1 5 1 R ( - ) -1,2-PDL 355 18 60 12 + S ( + ) -1,2-PDL 595 30 330 66 + ( RS ) -1,2-PDL 470 24 110 22 + a Cultures of JE1734 ( cob-24-4ac ) and RT818 ( pdu-8-4ac ) were grown in NCE-succinate-acetate minimal-medium containing magnesium ions , methi-onine , and the specified 1,2-PDL enantiomer under highly aerated conditions .
The,-galactosidase enzyme activity level was measured in early-log-phase cultures .
6 Growth on a specific enantiomer of 1,2-PDL was assessed in NCE medium containing magnesium ions , cobalamin , and the given enantiomer at a final concentration of 12 mM .
R ( - ) -1,2-PDL was used , and a 24-fold increase in transcription was measured with the racemic-mixture .
These results show a slight preference for S ( + ) -1,2-PDL as an effector molecule .
This apparent lack of specificity for either enanti-omer could be explained by the presence of an isomerase in the cell which would convert R ( - ) to S ( + ) -1,2-PDL or , alternatively , by a lack of specificity for either one of the enantiomers .
The latter is not unprecedented , since the 1,2-PDL dehydrase of Klebsiella pneumoniae catalyzes the dehydration of either enantiomer of 1,2-PDL , albeit at different rates ( 20 ) .
In our hands , S. typhimurium was able to grow aerobically on medium containing either of the enanti-omers as a carbon and energy source and cobalamin ( Table 3 ) .
Similarly to cobI regulation , the level of transcription of an operon fusion to the PDL utilization ( pdu ) ( 13 ) region was affected by both 1,2-PDL enantiomers ( Table ' 3 ) .
In this case , however , the effect of S ( + ) -1,2-PDL ( 66-fold increase ) was more pronounced than the one measured with R ( - ) -1,2-PDL ( 12-fold increase ) ; the racemate yielded an intermediate value of a 22-fold increase .
The differences in the induction level between cob and pdu in response to 1,2-PDL may reflect the involvement of other factors that at this point remain undetermined .
Carbon and energy source effects on the 1,2-PDL-dependent transcription of cobI .
Transcription of the cob-24-lac fusion in response to 1,2-PDL under aerobic-growth-conditions was clearly affected by the carbon and energy source present in the medium .
Increases in f-galactosidase activity in response to 1,2-PDL were measured when the cells were grown in minimal-medium containing a poor source of carbon .
We found that the expression of the fusion in cells grown on succinate was greater than that in cells grown on pyruvate , which was greater than that in cells grown on acetate ( data not shown ) .
Some experiments ( e.g. , Table 3 ) were performed using succinate with acetate as a supplement to increase the rate of growth of the cultures .
However , the cells grew very slowly anaerobically on this me-dium with nitrate as the alternative electron-acceptor .
Thus , for practical reasons subsequent culturing of strains under anoxic conditions was performed with a pyruvate-acetate medium .
Cells grown under more energetically favorable growth-conditions ( e.g. , minimal-medium with glucose ) also expressed cobI in response to 1,2-PDL ; however , the levels of induction were much lower than in minimal-medium with succinate 1,2-PDL-stimulated transcription of cobI does not result in de novo synthesis ofAdo-Cbi under aerobic-growth-conditions .
An S. typhimunum metE mutant which depends on coba-lamin for the synthesis of methionine from homocysteine ( 6 ) is prototrophic when grown anaerobically on cobalt-contain-ing minimal-medium .
This correction of the auxotrophic requirement for methionine is due to de novo synthesis of cobalamin , the coenzyme of methionine synthase ( MetH enzyme ) ( 14 ) .
Aerobically , however , even when 1,2-PDL was included in the medium , the metE strain remained auxotrophic for methionine .
In other words , despite the dramatic 1,2-PDL-dependent increase in cobI transcription under aerobic-growth-conditions , no net synthesis of the end product of the CobI pathway ( Ado-Cbi ) was observed .
This observation is not unprecedented , as a previous report by Andersson and Roth ( 1 ) showed that mutants with significant ( .80-fold increase ) aerobic transcription of cobI genes were still unable to synthesize cobalamin aerobically .
The 1,2-PDL-dependent control region .
The first indication of the location of the region that mediates the 1,2-PDL-dependent control of cob transcription was obtained from the following experiments .
We measured 0-galactosidase levels in strains carrying a TnlO insertion in CobI ( cob-ii : : TniO ) and a fusion in CobI ( cob-24-lac , JE102 ) , CobII ( cob-62-lac , JE100 ) , or CobIII ( cob-66-lac , JE101 ) ( 8 ) .
A strain carrying insertion cob-i1 : : Tn1O in an otherwise wild-type genetic background displayed a Cobl-phenotype .
The cob-ll : : TnJO insertion has been previously shown to have strong polar effects on the transcription of all the above-mentioned cob-lac fusions , and the polar effects were not correctible by a CobI + region provided in trans ( 8 ) .
As shown in Table 4 , insertion cob-1i : : TniO had strong polar effects on the 1,2-PDL-dependent expression of all three cob-lac fusions tested .
These data suggested that transcription from the PcobII and PcobJII promoters may not be regulated by the 1,2-PDL-dependent regulatory system , since one would expect no effect of the cob-ii : : TnIO insertion on the cobII-lac and cobIII-lac fusions if separate 1,2-PDL-dependent regulatory regions existed for each region of the operon .
These data also suggested that the increase in cobII and cobIII expression previously observed in response to 1,2-PDL was probably due to transcription initiation at the CobI promoter .
Isolation of mutants unresponsive to 1,2-PDL .
The isolation of mutants unable to regulate cobI transcription in response to 1,2-PDL was facilitated by two facts : ( i ) strain JE1734 ( cob-24-lac ) would grow with lactose as the sole carbon source only if 1,2-PDL was added to the medium , and ( ii ) colonies of JE1734 growing on minimal-medium containing X-Gal were white in the absence of 1,2-PDL and blue in its presence .
We used these phenotypes to screen a pool of mutant strains each carrying one insertion of the TnlOd ( Tc ) element in their genome .
Using lactose-containing medium , we screened approximately 10,000 Tcr transductants and found six mutants that were unable to grow on lactose ( Lac - ) even when 1,2-PDL was present in the medium .
The isolation of Lac-strains suggested the involvement of positive regulator mediating the effect of 1,2-PDL .
In this screen no mutants with the ability to grow on lactose without added 1,2-PDL were found ; i.e. , constitutive expression of cob was not observed .
Using the color phenotype on X-Gal-containing plates as a screen , we isolated 17 Tcr transductants ( from approximately 10,000 Tcr transductants analyzed ) with a colony color lighter than that of the wild type .
Again , the X-Gal-phenotype of these mutants was consistent with mutations that prevented the synthesis of some component of the 1,2-PDL-dependent positive regulatory system .
We also isolated strains with the appropriate phenotype ( Lac-X-Gal - ) but which were no longer kanamycin resistant .
We presumed that these strains no longer contained the fusion and did not study these strains further .
Mapping the TnlOd ( Tc ) elements .
Individual P22 phage lysates were grown on six of the strains ( Pdu-class ; see below ) containing the TnlOd ( Tc ) element in a TR6583 ( cob ' pdu + ) background and were used to transduce JE1734 ( cob-24-lac ) to Tcr .
This was done to reconstruct the mutant strains and to determine possible linkage of the insertions to cob-24-lac .
The insertions showed 65 to 75 % linkage to cob-24-lac .
When the insertions were moved into RT818 ( pdu-8-lac ) and tested for linkage , they showed 10 to 20 % linkage to the pdu-8-lac fusion .
These results suggested that the insertions were probably located between the cob and pdu regions , as the two regions are about 12 % linked by cotransduction ( 13 ) .
Cob and Pdu phenotypes of the mutants : the poc locus .
Several reasons prompted us to assess the CobI and Pdu phenotypes of the mutants carrying the TnlOd ( Tc ) elements .
( i ) The cob and pdu regions are closely linked , and they are divergently transcribed .
This is one feature of genes sharing common regulatory systems ( 5 ) .
( ii ) The expression of cob and pdu was positively regulated in response to 1,2-PDL .
( iii ) All of the TnlOd ( Tc ) elements affecting 1,2-PDL-depen-dent transcription of cob mapped in the region between cob and pdu .
The Cob and Pdu phenotypes of the isolated mutants were determined by mobilizing the TnlOd ( Tc ) elements into strain TR6583 ( cob ' pdu + ) by transduction .
The CobI phenotype of these strains was assessed under anaerobic-growth-conditions that demanded synthesis of Ado-Cbi .
The Pdu phenotype was assessed under aerobic-growth-conditions in medium containing 1,2-PDL as the sole carbon and energy source and cobalamin .
Three classes of mutants were identified on the basis of their Pdu and CobI phenotypes : ( i ) Pdu + Cobl-strains , ( ii ) Pdu-CobI + strains , and ( iii ) Pdu-CobI-strains .
Representatives of the two classes of Pdu-mutants were chosen for further study : JE1935 [ poc-102 : : Tn1Od ( Tc ) ] represents the Cob ' Pdu-class , and JE1939 [ poc-106 : : TnlOd ( Tc ) ] represents the Cob-Pdu-class .
- lac and pdu-lac fusions was found to be uninducible or marginally inducible by 1,2-PDL in strains containing an insertion in the poc locus ( Table 5 ) .
The effect of the poc-102 : : TnJOd ( Tc ) insertion on cob and pdu transcriptio poc-102 : : TnlOd ( Tc ) 25 80 + 5 100 poc-106 : : TnJOd ( Tc ) 10 15-5 5 poc ' ( JE1734 ) 30 310 + NA NA NT poc ' ( RT818 ) NA NA NT 5 715 + aCultures were grown on pyruvate-acetate minimal-medium plus nitrate and bicarbonate , with or without added 1,2-PDL .
Enzyme activity was determined in early-log phase cultures .
CobI and Pdu phenotypes were determined in strains containing only thepoc mutations .
The CobI phenotype was determined by the ability to grow anaerobically on plates containing glucose as the carbon source , without added cobinamide .
The Pdu phenotype was assessed in NCE medium containing magnesium ions , cobalamin , and 1,2-PDL .
NA , not applicable ; NT , not tested .
reproducibly different from the of the poc-106 : : was effect TnlOd ( Tc ) insertion .
In light of the fact that single mutation affected the a transcriptional regulation of both pdu and cobl , referred we the locus affected by these mutations the ( PDL and to as poc cobalamin ) locus .
Effect poc : : TnlOd ( Tc ) insertions the redox regula-of the on tion of cobl .
cobI expression previously shown to gene was be regulated by the redox level of the cell , such that expression is much higher anaerobically than aerobically ( 2 , 9 ) .
We investigated the effect of poc insertions the redox on regulation of cobl .
To do this compared the levels of we transcription of the previously studied cob-24-lac fusion in the absence of insertion in the chromopresence or a poc some .
The experiments performed in cultures were grown aerobically anaerobically in medium containing glycerol the or as carbon source and nitrate as the alternative electron-acceptor .
Glycerol was chosen as the carbon source , since growth on this medium best illustrates the redox control exerted on cobI expression ( 9 ) .
It is important to emphasize that in these experiments 1,2-PDL was not included in the culture medium .
As shown in Table 6 , cobI expression under anaerobic conditions was drastically reduced in poc mutants .
One implication from these data is that the poc gene product ( s ) may be involved in the redox control of cobl expression .
However , since the poc mutations tested were insertions , it is possible that their effect on redox control of poc-102 : : TnlOd ( Tc ) 5 60 poc-106 : : TnlOd ( Tc ) 5 15 poc ' 5 365 aAll strains are derivatives of JE1734 and carry the cob-24-lac operon fusion and the indicated poC allele .
Cells were grown aerobically on glycerol minimal-medium or anaerobically on the same medium plus nitrate and bicarbonate ; 1,2-PDL was not present in the medium .
Lacking poc ' Carrying poc + grown : grown : - PDL + PDL-PDL + PDL poc-102 : : TnlOd ( Tc ) 3 35 3 310 poc-106 : : TnlOd ( Tc ) 3 3 3 205 poc + 3 465 3 555 '' The strains tested are all derived from RT818 ( pdu-8-lac ) .
All strains carried a recAl allele .
Cultures were grown aerobically on succinate minimal-medium with or without 1,2-PDL .
1-Galactosidase activity was determined in early-log-phase cultures .
cobI expression was due to polarity effects on genes whose products are required for the redox control to be exerted or on a gene required for the expression of the poc locus itself ; insertion element or alternatively , it is possible that the somehow destabilizes the cob transcript .
An F ' plasmid carrying a wild-type copy of the region between cobI and pdu ( 1 ) was introduced into recombination-deficient ( recAl ) cob-24-lac poc : : TnlOd ( Tc ) strains and recAl pdu-8 : : Mu dl-8 poc : : TnlOd ( Tc ) strains to test for complementation of Poc function ( s ) .
We compared the response to 1,2-PDL in poc mutants carrying or lacking the F ' plasmid .
Table 7 presents only the data obtained in experiments using the pdu-8-lac fusion , but similar results were obtained with strains carrying the cob-24-lac fusion ( data not shown ) .
The levels of 3-galactosidase produced by poc mutant-F ' poc + merodipincreased 68-or 103-fold over loids in response to 1,2-PDL those in strains lacking the plasmid [ Table 7 , poc-106 : : TnlOd ( Tc ) andpoc + ] .
Complementation was also seen when the same strains were grown anaerobically ( data not shown ) .
These results suggest that the poc insertions disrupt the production of a diffusible factor and that the Poc phenotype is not due merely to disruption of the cob orpdu transcripts .
DISCUSSION The data presented in this communication suggest that 1,2-PDL is a positive effector of transcription of the coba-lamin biosynthetic ( cob ) and PDL utilization ( pdu ) genes of S. typhimurium .
Prior to this report only the transcription of the pdu genes was known to be affected by this diol ( 13 ) .
The stimulatory effects of 1,2-PDL on cob and pdu transcription can be clearly observed both under aerobic and anaerobic-growth-conditions and appear to be mediated by the gene product ( s ) of the newly defined poc ( PDL and cobalamin ) locus .
Mutations in poc render both pdu and cob unresponsive to the stimulatory effect of 1,2-PDL , suggesting that the poc gene product ( s ) alone is sufficient for the effect of 1,2-PDL to be exerted .
The idea thatpoc synthesizes one or more diffusible gene products is supported by the fact that mutations in poc are recessive to the wild-type allele when the latter is provided in trans .
We think that it is unlikely that a metabolite of 1,2-PDL , rather than 1,2-PDL itself , is the effector molecule for several reasons : ( i ) the products of the Pdu pathway , propionaldehyde and propionate , did not cause an increase in transcription of fusion cob-24 : : lac ( see above , `` Specificity for even though at least propionate is known to 1,2-PDL '' ) , serve as a carbon and energy source for S. typhimurium ( 11 ) ; and ( ii ) 1,2-PDL catabolism requires cobalamin , under aer obic conditions de novo synthesis of cobalamin does not occur in S. typhimurium , and cobalamin was never present in the culture medium in the experiments presented herein .
Furthermore , a strain carrying fusion cob-24-lac ( a strain unable to synthesize Ado-Cbi de novo ) was able to regulate the transcription of the fusion in response to 1,2-PDL under anaerobic conditions in minimal-medium lacking cobalamin ( Table 5 ) .
Since cobalamin is required for 1,2-PDL catabo-lism , it is unlikely that a breakdown metabolite rather than 1,2-PDL is the effector .
The genetic organization of thepdu and cob genes relative to one another is common to other loci whose transcription is controlled by a single regulatory system ( 5 ) .
In S. typhi-munum the pdu genes are located upstream and closely linked to the cob operon , and these regions are divergently transcribed .
We have mapped the poc locus to the region located between cob and pdu .
The coregulation of the cob and pdu genes intuitively makes sense , since the utilization of 1,2-PDL as a carbon and energy source by S. typhimurium requires the availability of adenosyl-cobalamin , the end product of the Cob pathway .
Even though our data clearly indicate that the cobI genes can be highly expressed under conditions of high oxygen levels , no net Ado-Cbi synthesis occurs under the conditions used in our experiments .
Since our data suggest that under aerobic conditions in the presence of 1,2-PDL , the entire cob operon is transcribed ( Table 5 ) , it is unclear why no net synthesis of Ado-Cbi is achieved .
One could suggest that failure to synthesize Ado-Cbi may be due to posttranscriptional regulation , to oxygen lability of Cob enzymes and/or intermediates of the CobI pathway , or to a combination of these or other unidentified factors .
It is interesting that under the laboratory conditions we have tested , utilization of 1,2-PDL as the carbon and energy source by S. typhimunum has been observed only under aerobic conditions , provided that cobalamin is present in the culture medium .
In this bacterium synthesis of cobalamin has not been observed in aerobically grown cells .
However , in the wild , S. typhimunum may encounter conditions of low oxygen levels where 1,2-PDL can be metabolized , and Ado-Cbi can be synthesized .
Another possible scenario would be one in which under anoxic conditions compounds such as rhamnose are catabolized and yield 1,2-PDL as an end product ( 16 ) .
The availability of the latter could then enhance the 1,2-PDL-dependent transcription of cob and pdu , resulting in adenosyl-cobalamin biosynthesis and the ability to further degrade 1,2-PDL to propanol and propion-ate as previously reported ( 16 ) .
This work was supported in part by the College of Agricultural and Life Sciences of the University of Wisconsin-Madison , by Public Health Service grant GM40313 from the National Institute of General Medical Sciences to J.C.E.-S. , and by National Science Foundation predoctoral fellowship RCD9154722 to M.R.R. .
We thank R. Jeter and J. R. Roth for providing strains , D. C. Cameron for the gift of 1,2-PDL enantiomers , and D. M. Downs for critical reading of the manuscript .
Our finding on the positive effect of 1,2-PDL on the aerobic transcription of the cob has been confirmed by Bobik genes et al. in the accompanying report ( T. A. Bobik , M. Ailion , and J. R. Roth , J. Bacteriol .
174:2253 -2266 , 1992 ) , and both groups have isolated mutants that are no longer responsive to 1,2-PDL .
In all instances , the lesions have been mapped to the region of the chromosome between cob and pdu .
Andersson , D. I. , and J. R. Roth .
Mutations affecting regulation of cobinamide biosynthesis in Salmonella typhimu-rium .
Andersson , D. I. , and J. R. Roth .
Redox regulation of the genes for cobinamide biosynthesis in Salmonella typhimunum .
Balch , W. E. , and R. S. Wolfe .
New approach to the cultivation of methanogenic bacteria : 2-mercaptoethanesulfonic acid ( HS-CoM ) - dependent growth of Methanobactenum ruminantium in a pressurized atmosphere .
Bassford , P. J. , Jr. , and R. J. Kadner .
Genetic analysis of components involved in vitamin-B12 uptake in Escherichia coli .
Beck , C. , and R. Warren .
Divergent promoters , a common form of gene organization .
Cohen , G. N. , and I. Saint-Girons .
Biosynthesis of threonine , lysine , and methionine , p. 429-444 .
In F. C. Neidhardt , J. L. Ingraham , K. B. Low , B. Magasanik , M. Schaechter , and E. Umbarger ( ed .
) , Eschenchia coli and Salmonella typhimunium : cellular and molecular biology .
American Society for Microbiology , Washington , D.C. 7 .
Davis , R. W. , D. Botstein , and J. R. Roth .
A manual for genetic engineering : advanced bacterial genetics .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 8 .
Escalante-Semerena , J. C. , M. G. Johnson , and J. R. Roth .
The CobIl and CoblIl regions of the cobalamin ( vitamin-B12 ) biosynthetic operon of Salmonella typhimurium .
Escalante-Semerena , J. C. , and J. R. Roth .
Regulation of cobalamin biosynthetic operons in Salmonella typhimurium .
Escalante-Semerena , J. C. , S.-J .
Suh , and J. R. Roth .
cobA function is required for both de novo cobalamin biosynthesis corrinoids in Salmonella typhiand assimilation of exogenous murium .
Gutnick , D. , J. M. Calvo , T. Klopotowski , and B. N. Ames .
Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimunium .
Heller , K. , and R. J. Kadner .
Nucleotide sequence of the gene for the vitamin-B12 receptor protein in the outer membrane of Eschenchia coli .
Cobalamin-dependent 1,2-propanediol utili-zation by Salmonella typhimurium .
Jeter , R. M. , B. M. Olivera , and J. R. Roth .
Salmonella typhimurium synthesizes cobalamin ( vitamin-B12 ) de novo under anaerobic-growth-conditions .
Experiments in molecular genetics .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 16 .
Obradors , N. , J. Badia , L. Baldoma , and J. Aguilar .
Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium .
Rondon , M. R. , and J. Escalante-Semerena .
Roof , D. M. , and J. R. Roth .
Ethanolamine utilization in Salmonella typhimunum .
A method for detection of phage mutants with altered transducing ability .
Schmieger , H. , and H. Bakhaus .
The origin of DNA in transducing particles of P22 mutants with increased transduction frequencies ( HT-mutants ) .
Toraya , T. , and S. Fukui .
In D. Dolphin ( ed .
John Wiley & Sons , Inc. , New York .
Way , J. C. , M. A. Davis , D. Morisato , D. E. Roberts , and N. Kleckner .
New TnlO derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition .