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Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118
Received 28 March 2002/ Accepted 16 May 2002
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ABSTRACT |
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TEXT |
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Since GlnR- and TnrA-regulated genes are expressed constitutively in glnA null mutants (13, 17), GS is required for the transduction of the nitrogen regulatory signal to TnrA and GlnR. It has been shown that feedback-inhibited GS forms a protein-protein complex with TnrA and that this interaction prevents TnrA from binding to DNA (18). Glutamine and AMP are the most effective feedback inhibitors of GS biosynthetic activity, while partial inhibition is observed with alanine, glycine, serine, and tryptophan (5). Mutations in TnrA that result in constitutive expression of the TnrA-activated amtB promoter all lie within the carboxy-terminal region of TnrA and impair the interaction between GS and TnrA (18). The mechanism by which GS regulates the activity of GlnR has not been elucidated.
Identification of the conditional glnRA mutations. Transcription of the amtB-glnK operon (formerly called nrgAB) is completely dependent upon TnrA (1, 16, 17). Along with J. M. Zalieckas, we previously described a procedure for isolating B. subtilis mutants with constitutive TnrA-dependent regulation (18). Mutants with high-level expression of an amtB-lacZ fusion were identified as blue colonies on glucose minimal plates that contained an excess nitrogen source and the chromogenic ß-galactosidase substrate 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal). Two of the mutants isolated in this screen had a conditional phenotype. These two mutants grew as white colonies when glutamine was used as the nitrogen source but formed blue colonies when either ammonium or glutamate plus ammonium was used as the nitrogen source. In contrast, wild-type cells grow as white colonies on X-Gal minimal plates containing any one of these three nitrogen sources. Genetic mapping revealed that both mutations responsible for this conditional amtB-lacZ phenotype were tightly linked to the glnA gene (18). For determination of the precise nucleotide lesion in these mutants, the glnRA operon from each mutant was PCR amplified and sequenced (18). One mutant was found to contain an insertion in the glnR coding region, while the sequence of the glnA ribosome binding site was altered in the other mutant.
Superrepressor phenotype of the glnR3 mutant. The glnR mutant contained an 8-bp insertion (5'-CGAAAAAA) located in the 3'-end of the glnR gene immediately after the codon for Lys-94. This insertion is a duplication of the 8 bp immediately preceding the insertion point. This allele, designated glnR3, produces a truncated GlnR protein in which the 41 carboxy-terminal amino acid residues are replaced by the tetrapeptide Arg-Lys-Asn-Gln.
The effect of the glnR3 mutation on the expression of the amtB-glnK operon was examined in cells containing an amtB-lacZ transcriptional fusion. When the cells were grown with either glutamine or glutamate as the nitrogen source, the levels of amtB-lacZ expression in the glnR3 mutant were similar to those in the wild-type strain (Table 1). In contrast, amtB was expressed at levels that were 820-fold higher in the glnR3 mutant than in the wild-type cells when the nitrogen source was glutamate plus ammonium (Table 1). These results confirm the conditional phenotype of the glnR3 mutant observed on plates and indicate that, unlike the case for wild-type cells, TnrA is transcriptionally active in glnR3 mutant cells grown with glutamate plus ammonium as the nitrogen source.
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GS has two roles in the regulation of the DNA-binding activity of TnrA. First, the enzymatic activity of GS is required for the synthesis of glutamine and other nitrogen-containing metabolites necessary for the formation of feedback-inhibited GS. Second, the feedback-inhibited form of the GS protein sequesters TnrA through a protein-protein interaction. Although the GS-dependent inhibition of TnrA activity is seen in glnR3 mutant cells grown with glutamine but not in glnR3 mutant cells grown with glutamate plus ammonium, similar levels of GS are present in glnR3 mutant cells under both growth conditions. This observation indicates that glnR3 mutant cells contain sufficient levels of GS protein to bind TnrA and block its DNA-binding activity. Thus, when glnR3 mutant cells are grown with glutamate plus ammonium, these cells must lack sufficiently high levels of glutamine and other GS feedback inhibitors to convert GS into its feedback-inhibited form. This conclusion is supported by observations indicating that growth of the glnR3 mutant is glutamine-limited compared with growth of wild-type cells in medium containing glutamate plus ammonium. First of all, the doubling time of glnR3 mutant cell cultures (83 min) is longer than that of wild-type cell cultures (60 min) with glutamate plus ammonium as the nitrogen source (Table 1). In contrast, glnR3 mutant and wild-type cells grow at similar rates with glutamine as the nitrogen source where no defect in TnrA regulation is observed in the glnR3 mutant cells (Table 1). Second, GS levels are twofold higher in wild-type cells grown with glutamate plus ammonium than in wild-type cells grown with glutamine, while the glnR3 mutant strain contains similar levels of GS in cells grown with either nitrogen source (Table 1). Taken together, these results indicate that the conditional TnrA phenotype of the glnR3 mutant is the indirect result of its inability to increase the synthesis of glutamine in response to nitrogen availability. As a result, when glnR3 mutant cells are grown in medium containing excess nitrogen but no glutamine, cell growth is nitrogen-limited and the intracellular levels of GS feedback inhibitors are never sufficient to convert GS to its feedback-inhibited form. No defect in TnrA regulation is observed in glutamine-grown glnR3 mutant cells because exogenous glutamine overcomes the defect in glutamine synthesis, allowing the formation of feedback-inhibited GS.
The glnA102 mutation lies within the glnA ribosome binding site. The glnA102 mutation was found to contain a base pair change in the ribosome binding site of the glnA gene (Fig. 1). Efficient translation initiation by B. subtilis ribosomes requires extensive complementarity between the mRNA ribosome binding site and the 3'-end of the 16S rRNA (3, 9, 15). Since GS levels in the glnA102 mutant are 7.5-fold lower than in wild-type cells (Table 2), the glnA102 mutation significantly reduces the translation efficiency of the glnA gene. The reduced level of GS present in the glnA102 mutant is sufficient to allow the formation of tiny colonies after 3 days of incubation at 37°C on glucose minimal medium plates containing glutamate plus ammonium as the nitrogen source. However, the glnA102 mutant could not be grown in liquid culture containing either glutamate or glutamate plus ammonium as the nitrogen source.
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The glnA102 and glnR3 mutants are similar in that both mutants synthesize reduced but significant levels of wild-type GS and have a conditional TnrA phenotype. This suggests that, as is the case for the glnR3 mutant, the reduced GS levels present in the glnA102 mutant are responsible for the altered TnrA regulation. One difference between the phenotypes of these two mutants is that glutamine-grown glnA102 mutant cells weakly activate amtB expression while amtB expression is completely repressed in the glnR3 mutant cells grown in this medium (Tables 1 and 2). Since GS levels are 6.5-fold lower in glutamine-grown glnA102 mutant cells than in the glnR3 mutant cells (Tables 1 and 2), this difference in the levels of GS protein and/or activity may be responsible for the low levels of amtB expression seen in the glnA102 mutant cells. Nonetheless, the observation that TnrA activity is almost completely inhibited in glutamine-grown glnA102 mutant cells, which have 7.5-fold lower GS levels than wild-type cells, indicates that the level of GS protein in a wild-type strain is significantly in excess of the amount needed to sequester and inactivate TnrA.
GlnR-dependent gene regulation in the glnA102 mutant was examined by use of a glnRA-lacZ fusion. Because TnrA represses expression of the glnRA operon by fourfold under nitrogen-limiting conditions (17), expression of the glnRA promoter was examined in a tnrA mutant background for these experiments. Expression of the glnRA promoter was completely derepressed in glutamine-grown glnA102 mutant cell cultures (Table 2). Although the GlnR-dependent repression of glnRA was completely relieved in the glnA102 mutant, only weak activation of the TnrA-dependent amtB promoter was observed. This observation may indicate that the regulation of GlnR activity by GS requires higher levels of GS than is needed for regulating TnrA activity. This difference would be expected if GlnR activity were regulated by a protein-protein interaction with GS and the amount of GlnR in the cell were higher than the amount of TnrA. Alternatively, GS-dependent regulation of GlnR activity may be more sensitive to changes in nitrogen availability than is the interaction between GS and TnrA. According to this hypothesis, growth of the glnA102 mutant cells in glutamine medium would be expected to be more nitrogen-limited than that of glnR3 mutant cells.
TnrA regulation in other Gln- mutants. The nucleotide lesions in several previously isolated glutamine-requiring mutants were also determined (6, 8). The leaky glnA93 mutant was found to contain the same base pair alteration in the glnA ribosome binding site as the glnA102 mutant (Fig. 1). The results from previous studies that determined the GS levels and effect of the glnA93 mutation on the expression of the glnRA and amtB promoters are in good agreement with the results obtained with the glnA102 mutant (1, 6, 13).
Three other Gln- mutants, glnA22, glnA70, and glnA73, require glutamine for growth on all nitrogen sources and contain undetectable levels of GS activity, i.e., 
3% of the GS levels seen in wild-type cells (6, 8). Expression of the amtB-lacZ and glnRA-lacZ fusions has been shown to be completely derepressed in strains containing the glnA73, glnA70, and glnA22 mutations (1, 13). The glnA73 mutant contains a G-to-A transition mutation within the glnA ribosome binding site in a nucleotide that is adjacent to the location of the glnA93 and glnA102 mutations (Fig. 1). Since there is no measurable GS enzymatic activity in the glnA73 mutant (8), this mutation severely impairs translation of the glnA gene. The glnA22 and glnA70 mutants contain the same C-to-A transversion that converts the UCA codon for Ser-249 into an ochre codon. This mutation would be expected to result in the synthesis of a truncated GS protein that lacked enzymatic activity. Thus, the lack of significant levels of wild-type GS protein is primarily responsible for the constitutive expression of TnrA-regulated genes observed in these Gln- mutants. Although the amino acid sequence of the GS protein is not altered in the glnA73 mutant, the level of GS protein present in the glnA73 mutant is insufficient to partially inhibit TnrA activity even when the glnA73 mutant is grown with glutamine as a nitrogen source.
Conclusions. For the regulatory interaction between GS and TnrA to occur, B. subtilis cells must contain adequate levels of the GS protein as well as the products of GS enzymatic activity, i.e., feedback inhibitors. The phenotypes of the glnRA mutants reported here are consistent with this model. Three mutants, the glnR3, glnA93, and glnA102 mutants, have a leaky Gln- phenotype due to the synthesis of reduced but significant levels of wild-type GS protein. GS inhibition of TnrA activity is seen in these mutants only during growth in medium containing glutamine, the product of GS enzymatic activity. In contrast, the Gln- mutants with undetectable levels of GS activity, i.e., the glnA73, glnA22, and glnA70 mutants, lack sufficient levels of wild-type GS protein to inhibit TnrA even when supplied with exogenous glutamine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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