Regulation by the glnR and glnF proteins subject to nitrogen control .
Part of the evidence for their proposal is that mutations that result in fixed , low-level synthesis of proteins under nitrogen control ( which they called the GlnR phenotype ) were found to lie within ginA ( 5 ) , as were mutations to constitutive high-level synthesis of the same proteins ( GlnC phenotype ) ( 6-9 ) .
Location of such mutations within glnA was based on three-factor transductional crosses and on complementation analysis .
Here we present evidence that a separate regulatory gene , glnR , lies very close to glnA and that mutations to complete loss of function of the glnR product result in the GlnR phenotype .
We propose that all mutations that result in the GlnR ( or GlnC ) phenotypes may lie within glnR rather than ginA .
ABSTRACT The product of the glnR gene is required for nitrogen regulation of the synthesis of glutamine synthetase ( Gln synthetase ) [ L-glutamate : ammonia ligase ( ADP-forming ) , EC 6.3.1.21 and two periplasmic transport proteins that are subject to nitrogen control in Salnonella .
Strains with mutations to loss of function of the glnR product [ e.g. , a strain with a TnlO insertion or one with an ICR-induced ( frameshift ) mutation in glnRJ have about 3 % as much Gln synthetase as a fully derepressed wild-type strain and are unable to increase synthesis of this enzyme or periplasmic transport proteins in response to nitrogen-limitation .
The structural gene for Gin synthetase , ginA , and those for the periplasmic transport proteins are unlinked on the chromosome ; thus , glnR appears to encode a diffusible positive regulatory element .
Consistent with this , the mutant glnR allele is recessive to the wild-type allele with regard to expression of glnA ( synthesis of Gln synthetase ) .
Although glnR is closely linked to ginA , strains with mutations to complete loss of function of the glnR product can be distinguished from ginA strains by their ability to produce detectable Gln synthetase and to grow in the absence of glutamine .
To demonstrate unequivocally that glnR is distinct from ginA , we have purified and characterized Gln synthetase from with TnlO a strain a insertion in glnR .
Because the properties of Gln synthetase from the insertion mutant , most importantly the carboxyl-terminal sequence of amino-acids , are the same as those of synthetase from wild type , the TnlO insertion can not be in ginA ( if it were , the carboxyl terminus of Gln synthetase would have to be altered ) ; therefore we conclude that the TnlO insertion is in a regulatory gene , glnR , which is distinct from ginA .
A model for functhe tion of the glnR product together with the previously defined glnF product in mediating nitrogen control is discussed .
In enteric bacteria , synthesis of glutamine synthetase ( Gln synthetase ) [ L-glutamate : ammonia ligase ( ADP-forming ) , EC 6.3.1.21 is controlled by availability of nitrogen in the growth medium ( 1 ) .
We previously demonstrated that the product of a positive regulatory gene , glnF , is essential for synthesis of this enzyme in Salmonella ( 2 ) .
We now have evidence that the product of a second positive regulatory gene , glnR , is also required .
In addition , both the glnR and glnF products appear to be required for nitrogen regulation of the synthesis of two periplasmic transport proteins ( a glutamine-binding protein and the arginine/lysine/ornithine-binding protein ) and , thus , they have pleiotropic effects .
Genetic studies indicate that the products of the glnR and glnF genes function together to mediate nitrogen control .
Magasanik , Tyler , and their colleagues have proposed that Gin synthetase mediates nitrogen control in Klebsiella aerogenes and Escherichia coli ( reviewed in refs .
Specifically , they propose that Gln synthetase functions directly as a genetic regulatory element to control transcription of its own structural gene , glnA ( autogenous regulation ) , and that it plays a major role in controlling transcription of genes for other The publication costs of this article were defrayed in part by page charge payment .
This article must therefore be hereby marked `` advertisement '' in accordance with 18 U. S. C. § 1734 solely to indicate this fact .
MATERIALS AND METHODS RESULTS SK292 ( glnR + / F'glnR + ) 0.37 SK215 ( glnR129 ) 0.04 SK217 ( glnR130 ) 0.04 TA831 ( wild type ) 0.22 SK178 ( glnAAR60/F ` glnR + ) 0.10 SK35 ( glnAAR60 ) < 0.01 * All strains contain the hisF645 mutation .
Strains carrying F'glnR + ( F ' 133 coli ) also contain the argHl823 : : TnlO mutation , which is covered by this episome .
t Not corrected for blank activity .
Strains were grown on medium E plus ( 13 ) 3 mM glutamine ( nitrogen-excess conditions ) .
Mapping and Complementation of g ~ nR .
Four glnR mutations that were studied extensively were 66-83 % linked by P22-mediated transduction to the glnA188 mutation ; the linkage of glnR137 : : TnlO was 71 % .
Complementation of glnRmutations with the E. coli episome F ' 133 suggested that the wild-type allele was dominant and , therefore , that glnR encodes a diffusible product , consistent with its having a pleiotropic role in nitrogen regulation .
A glnRI - / glnR + merodiploid ( lines 1 and 2 , Table 2 ) has as much Gln synthetase activity as a glnR + / glnR + strain ( line 3 ) and considerably more than the sum of the activities ( 0.14 ) expected if the chromosome ( lines 4 and 5 ) and the episome ( line 7 ) were functioning independently .
( Independent function would have been expected if glnR were a cis-acting regulatory site that controlled expression of the adjacent glnA gene .
) Properties of Gln Synthetase from SK398 ( glnR137 : : TnlO ) .
The growth behavior and Gln synthetase specific activities of glnR strains ( Table 1 ) distinguish them from ginA strains .
To demonstrate unequivocally that ginR is distinct from ginA , we purified Gln synthetase ( Table 3 ) from strain SK398 ( glnR137 : : TnlO ) and compared its properties with those of the synthetase from strain TA831 ( wild type ) .
Recoveries of activity suggested that GIn synthetase from the mutant was stable to heat , acetone , and acid-pH , as was the enzyme from wild type .
Consistent with the relative specific activities in extracts of Table 3 .
Purification of Gln synthetase from strains SK398 ( glnR137 : : TnlO ) and TA831 ( wild type ) Specific activity , , mol/min-mg Recovery , % TA831 SK398 TA831 SK398 0.19 0.03 100 100 0.67 0.09 97 60 2.3 0.18 119 63 6.0 1.3 98 56 13.9 3.1 76 46 57.2 47.8 53 36 Fraction Crude * ( NH4 ) 2S04 ppt .
Heated supernatant Acetone ppt .
Affi-Gel Blue eluatet P'hosphodiesterasetreatedt 48.0 50.1 l'otal units were 3645 for TA831 and 2937 for SK398 .
t Samples were applied in the presence of 10 mM glutamate , and Gln synthetase was eluted in the presence of 10 mM glutamate , 0.1 M KCI , and 10 mM ADP .
As described in Materials and Methods .
The reaction was complete in 2 hr .
The ratio of activities + Mg2 + / - Mg2 + ( 18 ) for Gln synthetase from TA831 increased from 0.5 to 1.4 after phosphodiesterase treatment , indicating that the synthetase was partially adenylylated ( 21 ) , whereas the ratio for strain SK398 was 1.4 and did not change .
Adenylylated Gin synthetase is the less active form ( 18 ) .
DISCUSSION lyzes synthesis of a low molecular weight coregulator of tran-scription-a signal of nitrogen deficiency ( Fig. 2 ) .
When bound to the low molecular weight signal , the glnR product would function as an activator of transcription for genes subject to nitrogen control ; in the absence of the nitrogen-deficiency signal the glnR product would function as an inhibitor ( repressor ) of transcription , at least for ginA .
( Note that a model in which glnR encodes the macromolecular regulator of transcription accounts for the fact that the properties of glnR glnF strains are the same as those of glnR strains .
) Magasanik , Tyler , and their colleagues have proposed that nitrogen regulation is mediated directly by Gin synthetase ( reviewed in refs .
3 and 4 ) and that the function of the enzyme as a regulator of transcription depends on its state of covalent modification ( 3 ) .
Several lines of evidence were presented in support of this model .
First , mutations that were reported to alter the degree of covalent modification of Gln synthetase had a major effect on synthesis of Gln synthetase and other proteins under nitrogen control in Klebsiella ( 3 ) .
We were unable to confirm this finding in Salmonella ( 21 ) .
Second , mutations that caused the GlnR and the GlnC phenotypes in Klebsiella ( see introduction ) were reported to lie within ginA , the structural gene for Gin synthetase .
Our data indicate that there is a sep-glnR , ginA the Salmonella chromo-arate gene , very near on some and that mutations to loss of function of glnR result in the GlnR phenotype .
Because the glnF gene has been identified in Klebsiella ( 5 ) , it is likely that there is also a glnR gene in this organism .
We think that mutations-causing the GlnR phenotype , which were isolated as suppressors of glnF ( 5 ) , may lie within glnR and not glnA .
Note Added in Proof .
Effects of the glnR product on expression of transport genes appear to be direct .
Newly isolated mutations near ginA ( presumably in a cis-acting site ginI ) result in high-level constitutive synthesis of Gln synthetase in a glnR-background ; these mutations do not , however , restore high-level synthesis of the glutamine-or ar-ginine/lysine/ornithine-binding proteins .
Thus , regulatory effects of the glnR product on synthesis of binding proteins do not appear to be mediated through Gln synthetase .
We thank K. Kanagaki for amino-acid analyses and G. F.-L .
Ames , S. W. Artz , L. N. Csonka , and J. L. Ingraham for thoughtful criticisms of the manuscript .
This work was supported by U.S. Public Health Service Grant GM 21307 .