Nitrogen regulatory locus `` glnR '' of enteric bacteria is composed cistrons ntrB and ntrC : Identification of their protein products ( regulation of gene expression/positive and negative control/nitrogen utilization/glutamine synthetase/in-vitro protein synthesis ) ABSTRACT The nitrogen regulatory locus `` glnR '' of Escher-ichia coli and Salmonella typhimurium is composed of two cistrons , which we propose to call ntrB and ntrC ( nitrogen regulation B and C ) .
Frameshift mutations in ntrB and ntrC were isolated on a A phage that carries the E. coli ntrB and ntrC genes and the closely linked ginA gene , the structural gene encoding glutamine synthe-tase [ L-glutamate : ammonia ligase ( ADP-forming ) , EC 6.3.1.2 ] ; mutations were selected as suppressors of glnF ( which we propose to rename ntrA ) , a selection used previously to isolate glnR mutations .
Phage DNA from one mutant ( ntrB ) failed to direct synthesis of a 36-kilodalton ( kDal ) protein whose synthesis was directed by DNA from the parent phage ( ntrB + ) in a coupled in-vitro-transcription/translation system .
DNA from three other mutants ( ntrC ) failed to direct synthesis of a 54-kDal protein ; DNA from two ofthese mutants instead directed synthesis of smaller proteins , 53 and 50 .
all four from In cases , DNA frame-shift revertants directed synthesis of both the 36-kDal and 54-kDal proteins .
These results suggested that ` ntrB and ntrC were separate genes which encoded 36-kDal and 54-kDal protein products , respectively .
Frameshift mutations in ntrB and ntrC complemented each other with regard to regulation of glnA expression in-vivo and growth on arginine as nitrogen source , another nitro-gen-controlled phenotype ; this confirmed that ntrB and ntrC are separate cistrons that encode diffusible products .
The ntrB and ntrC genes were also defined in S. typhimurium .
Studies of mutant strains provided information on the roles of the ntrB and ntrC products in activation and repression ofginA expression and raised-the possibility that these products function as a protein complex in regulating expression of nitrogen-controlled genes .
bacteria , In enteric synthesis of several proteins including glutamine synthetase [ Gln synthetase ; L-glutamate : ammonia ligase ( ADP-forming ) , EC 6.3.1.2 ] is controlled by availability of nitrogen in the growth medium ; synthesis of these proteins is increased under nitrogen-limiting conditions ( 1 , 2 ) ( reviewed in ref .
of two positive regulatory genes , glnF and ginR [ called ginG Escherichia ( 4 ) ] in coli are required for ni-control ( 5 , 6 ) .
loss glnF trogen Mutations to of function of and several previously characterized loss mutations to of function of glnR result in loss of ability to express nitrogen-controlled genes at high levels ( 5 ) .
We have proposed the following working model for the functions of the glnF and glnR products ( 5 ) ( Fig. 1 ) .
The glnF product converts the glnR product to a form with positive regulatory character .
By analogy with other bacterial regulatory mechanisms , the glnF product may catalyze synthesis of a low molecular weight signal of nitrogen-limitation which binds to the glnR product , or the glnF product may interact directly with the glnR product .
In addition to having positive regulatory character , the glnR product has negative regulatory character ( 5 ) .
This was inferred because mutations to loss of function of glnR suppress the glutamine requirement caused by mutations to loss of function of glnF .
That is , glnR-glnF-strains make more Gln synthetase than do glnF-strains and can grow in the absence of glutamine .
Again by analogy with other bacterial regulatory mechanisms , the glnR product may be a macromolecular regulator of transcription which exists in two forms-one that represses transcription of glnA , the structural gene for Gln synthetase , and one that activates transcription of glnA and other genes subject to nitrogen control [ in Salmonella typhimurium these include genes for amino-acid transport components ( 7 ) ] .
The glnF product would lead to formation of the activator form .
In a glnFbackground the glnR product would have exclusively negative regulatory character .
We have now found that mutations at the locus `` ginR '' which suppress glnF lie in two cistrons rather than one .
We have identified the protein products of these cistrons and have studied their individual functions in nitrogen regulation in-vivo .
Because the `` glnR gene '' consists of two cistrons , nomenclature for this gene must be revised .
We propose to as rename ginF ntrA and `` glnR '' as ntrB and ntrC because these genes have a pleiotropic role in nitrogen regulation which is not restricted to control of the glnA gene ( ntr for nitrogen regulation ) .
MATERIALS AND METHODS Specialized A Transducing Phages .
Seven AglnA transducing phages carrying the E. coli ginA region ( AglnA1-AglnA7 ) isolated by the technique of Shrenk and Weisberg ( 8 ) were obtained from James Friesen .
Physical characterization of the phages indicated that each carried a different amount of bacterial DNA adjacent.to glnA ( unpublished data ) .
To determine by complementation .
analysis whether AglnA phages carried `` glnR '' ( ntrB and ntrC ) and to isolate `` glnR '' mutations on them , they were transferred to well-characterized S. typhimurium mutant strains ( 5 , 6 ) .
For transfer they were first lysogenized on an E. coli F'gal episome ( F ' 100-12 ) ( 9 ) by infecting E. coli strain NCM79 ( glnA3 recA56 galT23 argA22 srl300 : : TnlO/F ' 100-12 ) with a heat-induced lysate of strain NCM6 [ glnA3 trpA9825 ( A c1857 S7 ; A cI857 S7 glnA6 ) ] or a similar double lysogen and selecting Gln + transductants .
Episomes carrying AglnA phages were then transferred to S. typhimurium strain SK72 [ A ( glnA-ntrB ) 60 * hisF645 galE1794 ] by selecting Gal ' transconjugants and screening for those that were also Gln + .
Subsequent transfers were made intragenerically .
In all cases , segregation of episomes carrying transducing phages was demonstrated .
Isolation of Mutant Strains .
Salmonella ntrB and ntrC mutations were isolated spontaneously by suppression of the glutamine requirement of strain SKlOO carrying ntrA76 ( 5 ) .
E. coli ntrB and ntrC mutations were isolated on AglnA6 by suppression of ntrA after mutagenesis ( with 2-chloro-6-methoxy-9 - [ 3 - ( 2-chloroethyl ) aminopropylamino ] acridine dihydrochloride [ ICR ] ) of S. typhimurnium strain SK720 [ ntrA75 A ( glnA-ntrB ) 60 galE1823 metB869 : : TnlO/F ' 100-12 ( A c1857 S7 ; A cI857 S7 , glnA6 ) ] .
ICR-induced revertants were also isolated .
Because A does not grow in S. typhimurium ( 10 ) , episomes carrying phages were transferred to an E. coli A lysogen ( NCM107 ) in order to isolate DNA template for use in-vitro .
Complementation in E. coli .
To perform intrageneric complementation analysis of E. coli ntrB and ntrC mutations , such mutations were initially recombined into the E. coli chromosome .
To do this , lysates were prepared from E. coli AglnA6 lysogens carrying ntrB or ntrC mutations on the phage .
The ntrB and ntrC mutations were recombined into the chromosome ofE .
coli strain NCM 155 ( glnA3 galT23 ) by selecting Gln + transductants at 420C and screening for those which were Aut - ( unable to grow on arginine as nitrogen source ) ( 5 ) and Aksensitive .
The Aut-phenotype ( ntrB or ntrC lesion ) was linked by P1-mediated transduction to a TnlO element inserted near glnA ( obtained from S. Kushner ) .
For complementation analysis , these strains were made recA ( 11 ) , and AglnA6 carrying an ntrB or ntrC mutation was introduced ( from an E. coli strain ) on F ' 100-12 .
Gal ' transconjugants were scored for Aut phenotype and resistance to A.Complementing strains were Aut + and A resistant .
Noncomplementing strains were A resistant but Aut - ; these strains were shown to contain AglnA6 by their ability to produce glnA ' transducing phages after heat induction .
Cells were grown to stationary-phase at 300C and cell extracts were prepared as described ( 12 ) in 10 mM imidazole , pH 7.3 / 1 mM MnCl2 .
Total activity of Gln synthetase was assayed as described ( 13 , 14 ) .
E. coli Gln synthetase was also assayed by using the Mn2I triethanolamine-dimethyl-glutaric acid assay mixture ( pH 7.57 ) of Stadtman et-al .
Levels of Gln synthetase antigen were determined as described ( 7 ) .
In Vitro Protein Synthesis .
Template DNA was extracted from AglnA phages purified after heat induction of coli double E. lysogens ( 16 ) .
DNA was used at a concentration of 100-200 jig / * Previously called A ( glnA-glnR ) 60 ( 5 ) .
This deletion apparently extends into ntrB because it fails to recombine with ntrB point mutations ; it does recombine with all ntrC point mutations that have we tested and therefore may or may not extend into ntrC .
The deletion was phenotypically NtrC-with respect to isolation of mutations on AglnA6 .
Standard conditions for coupled transcription/translation assays have been described ( 17 ) .
Assays were done at optimal concentrations of amino-acids , tRNA , Mg2 + , K + , and S-30 ( to be described elsewhere ) .
S-30 cell extracts were prepared from S. typhimurium strain SK417 ( ntrA76 relAl hisT1504 hisA2253 ) or SK416 [ A ( glnA-ntrB ) 60 zig-205 : : TnlO relAl hisT1504 hisA2253 ] .
Proteins synthesized in-vitro were labeled with [ 3S ] methionine ( 5 , uCi per reaction ; 1 Ci = 3.7 x 1010 becque-rels ) and were separated by sodium dodecyl sulfate/polyacryl-amide gel electrophoresis ( 18 ) .
Gels were fixed , stained ( 19 ) , dried , and placed under film ( Kodak X-Omat XR-2 ) .
The frameshift mutagen ICR 191E was kindly donated by H. J. Creech .
L - [ 3S ] Methionine ( 1017.8 Ci/mmol ) and '' ` C-labeled molecular weight standards were obtained from New England Nuclear .
RESULTS Characterization of AglnA Phages for the Presence of the `` glnR '' Locus by Complementation Analysis .
Seven different A transducing phages that carry the E. coli glnA region were characterized by complementation analysis for presence of the `` glnR gene .
'' Phages were transferred to S. typhimurium recipients containing a chromosomal deletion of part of the glnA - `` glnRP region ( see footnote * ) .
All merodiploids were glutamine independent , confirming that all seven phages carried an intact ginA gene .
We will refer to these phages as AglnA1-AglnA7 .
A merodiploid carrying one of these phages , AglnA2 , failed to utilize arginine as nitrogen source , indicating that AglnA2 lacked a functional `` glnR gene '' ( 5 ) .
Table 1 summarizes Gln synthetase activities for several of the S. typhimurium strains harboring AglnA phages .
Strain SK595 carrying AglnA6 ( `` glnR + '' ) was able to increase synthesis of Gln synthetase approximately 30-fold in response to nitrogen-limitation , similar to a control strain ( SK726 ) carrying the E. coli episome F ' 133 , which covers the ginA region .
The presence of a chromosomal ntrA mutation in such strains resulted in synthesis of low , unregulated levels of Gln synthetase ( strains SK727 and SK588 ) .
In contrast to the above `` glnR + '' merodip-loids , strain SK574 carrying AglnA2 ( `` glnR '' ) was unable to synthesize high levels of Gln synthetase under nitrogen-limiting conditions .
The low level of synthesis in this strain was independent of ntrA ( see strain SK584 ) as expected based on previous studies of S. typhimurium `` glnR '' mutant strains ( 5 ) .
Activation of synthesis of Gln synthetase from AglnA2 was restored in a merodiploid with a functional `` glnR '' locus on the chromosome ( strain SK801 ) and was dependent on a functional ntrA gene ( strain SK834 ) .
These results confirm that the `` glnR product '' works in trans and therefore is diffusible ( 5 ) .
They also indicate that cis-acting regulatory sites adjacent to ginA on AglnA2 are intact .
Identification of Two Protein Products of the `` glnR '' Locus ( ntrB and ntrC ) .
When DNA from AglnA6 was used as template in a coupled in-vitro * transcription/translation system , it directed synthesis of six major bacterial proteins [ 75 , 68.5 , 56 , 54 , 47.5 , and 36 kilodaltons ( kDal ) , ` respectively ] ( Fig. 2 , lane 2 ) the of the gene , was identified ( Gln synthetase , product ginA with antiserum as the 56-kDal protein ) .
by precipitation specific DNA from AglnA2 , which was `` glnR '' as judged by complementation analysis , failed to direct synthesis of the 54-and 36-kDal proteins and of a 47.5-kDal protein ( Fig. 2 , lane 1 ) .
Synthesis of the 54-and 36-kDal proteins was directed by six inincluding AglnA6 .
The dependent AglnA + `` glnR + '' phages , 47.5-kDal protein was not correlated with the presence or absence of a functional `` glnR '' locus because several AglnA + `` glnR + '' phages as well as AglnA2 failed to direct its synthesis To determine whether either the 54-kDal or 36-1kDal protein was the `` glnR '' product , we isolated `` glnR '' fram eshift mutations on AglnA6 .
DNA from one such mutant ( ntriB1 ) failed to direct synthesis of the 36-kDal protein in-vitro ( Figg .
DNA from three other mutants ( ntrC3 , ntrC4 , and rtrC5 ) failed to direct synthesis of the 54-kDal protein ( Fig. 2 , laines 5 , 7 , and 9 ) ; DNA from two of these mutants ( ntrC4 and ntirC5 ) instead directed synthesis of smaller proteins , 53 and 50 k ] Dal , respectively ( presumably , premature termination products ) .
In all cases , DNA from frameshift revertants directed synthesis of both the 36-kDal and 54-kDal proteins ( Fig. 2 ; lanes 4 , 6 , 8 , and 10 ) .
DNA from both frameshift mutants and revertants directed synthesis of all other proteins , including Gln synthetase , whose synthesis was directed by DNA from AglnA6 .
These results indicated that the `` glnR gene '' ( 5 ) might , in fact , be two genes ( ntrB and ntrC ) that encoded 36-kDal and 54-kDal protein products , respectively .
Complementation Analysis of E. coli ntrB and ntrC Mutations .
ICR-induced mutations ntrBI , ntrB2 , ntrC3 , and ntrC4 isolated on AglnA6 were recombined into the E. coli chromosome and analyzed for their effects on synthesis of Gln synthe-tase ( Table 2 .
) Strain NCM67 ( ntrB + ntrC + ) increased synthesis of Gln synthetase 10-fold under N-limiting conditions .
The two ntrC strains ( NCM 131 and -133 ) produced low levels of Gln synthetase and failed to increase its synthesis under N-limiting conditions .
The two ntrB strains ( NCM 130 and -134 ) produced high levels of Gln synthetase even when grown with a high concentration of ammonia .
Merodiploid strains with an ntrB mutation on the chromosome and an ntrC mutation on AglnA6 ( or vice versa ) regained the ability to regulate synthesis of Gln synthetase over about a 10-fold range-in response to availability of ammonia ( Table 2 , strains NCM 147 , -145 , -150 , and -152 ) and , to utilize arginine as nitrogen source .
Control strains carrying ntrB or ntrC mutations on both chromosome and A did not regain the ability to regulate synthesis of Gln synthetase ( strains NCM 149 , -151 , -146 , and -148 ) or grow on arginine .
Thus , mutations in ntrB and ntrC complement each other , indicating that ntrB and ntrC are separate cistrons that encode diffusible products and that both products are required for what had previously been defined as `` glnR '' function .
kglnA2 , which was `` glnR - '' and failed to direct synthesis of both the 54-kDal and 36-kDal proteins , was demonstrated to lack both ntrB and ntrC function genetically because it failed to complement either the ntrBI or ntrC4 mutation on the E. coli chromosome for growth on arginine as nitrogen source ( data n t s ) nooshhowwn ) .
Analysis of S. typhimurium ntrB and ntrC Mutations .
Having isolated E. coli ntrB and ntrC mutations , we wanted to determine whether similar mutations occurred in S. typhimurium .
We therefore studied a number of spontaneous ntrA suppressors in S. typhimurium .
These were crossed into an ntrA + back Strain Relevant genotype N excess N limiting TA831 ntrB + ntrC + 0.13 1.8 SK639 ntrC302 0.03 0.05 SK652 ntrC315 0.02 0.06 SK611 ntrB243 1.2 2.7 SK622 ntrB285 1.2 2.6 SK100 ntrA76 < 0.01 < 0.01 SK517 ntrA76 ntrC302 0.02 0.05 SK530 ntrA76 ntrC315 0.02 0.06 SK488 ntrA76 ntrB243 0.03 0.06-SK500 ntrA76 ntrB285 0.03 0.06 - * Levels ofGln synthetase activity and antigen were correlated .
Media were as in Table 1 .
t Ability to use arginine as sole nitrogen source .
DISCUSSION The previously defined nitrogen regulatory locus ( `` ginR '' ) of E. coli and S. typhimurium is not a single gene but is composed of-two cistrons , ntrB and ntrC .
The products ofthese genes have been identified as a 36-kDal protein and a 54-kDal protein , respectively .
The ntrB and ntrC genes are closely linked to each other and to ginA in both S. typhimurium and E. coli .
In S. typhimurium , the order of genes in this region is polA ntrC ntrB ginA rha ( unpublished data ) .
A loss-of-function mutation in either ntrB or ntrC suppresses the glutamine requirement caused by ntrA ( glnF ) mutations .
Suppression of ntrA by ntrB or ntrC mutations is presumably due to loss of negative regulation of ginA expression by these gene products ( Fig. 1 ; Table 3 ; ref .
Double-mutant strains ntrB ntrA and ntrC ntrA synthesize Gln synthetase at low levels ; apparently , ginA expression can be neither repressed nor activated-in these strains .
In an ntrA + background , ntrB and ntrC mutations affect ginA expression differently .
An ntrC strain , which lacks the 54-kDal protein , appears to have no residual nitrogen regulatory function ( 5 ) .
It neither activates nor represses ginA expression-Gln synthetase is synthesized at low levels .
An ntrB strain , which lacks the 36-kDal protein , retains ability to activate ginA expression fully and , in fact , synthesizes Gln synthetase at high levels in a defined minimal-medium containing a high concentration of ammonia ( sufficient to decrease synthesis in a wild-type strain ) ( Tables 2 and 3 ) .
From these results we infer that the ntrC product ( 54-kDal protein ) without the ntrB product ( 36-kDal protein ) is sufficient to mediate ntrA-dependent activation of ginA expression .
Both the ntrB and ntrC products appear to be required for negative regulation of ginA expression .
A working model that can account for participation of the ntrA , ntrB , and ntrC products in regulating ginA expressio and that ofother nitrogen-controlled genes ( 5 ) is outlined in Fig. 1 .
It is clear that ntrB and ntrC mutations alter expression of nitrogen-controlled genes .
An attractive hypothesis is that the ntrB and ntrC products function as a protein complex and act directly the level of at transcription .
We are grateful to Dr. James Friesen for sending us the AglnA phages , M. K. McKinley for expert assistance in performing some of the complementation studies , and G. F.-L .
Ames , B. Bochner , L. N. Csonka , and J. L. Ingraham for thoughtful criticisms of the manuscript .
This work was supported by U.S. Public Health Service Grants GM21307 and GM27307 to S.K. and S.A. , respectively .
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