Genetic and biochemical analysis of phosphatase activity of Escherichia coli NRII (NtrB) and its regulation by the PII signal transduction protein

Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.     

Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli.

The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved.     

Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli.

DNA was prepared from a strain of Escherichia coli bearing a mutation which confers the GlnC phenotype (inability to reduce the expression of glnA and other nitrogen-regulated operons in response to ammonia in the growth medium). A fragment of this DNA carrying glnA, the structural gene for glutamine synthetase, was cloned on plasmid pBR322. By using recombination in vitro, we mapped the GlnC mutation to a region between glnA and glnG. This region defines a gene, glnL, which codes for a trans-acting product; the GlnC mutant produces an altered product. The glnL product plays a key role in the communication of information concerning the quality and abundance of the nitrogen source in the growth medium to a destination responsible for the regulation of glnA and other genes for enzymes responsible for nitrogen utilization.     

Mutational analysis of the bacterial signal-transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB).

The signal-transducing kinase/phosphatase nitrogen regulator II (NRII or NtrB) is required for the efficient positive and negative regulation of glnA, encoding glutamine synthetase, and the Ntr regulon in response to the availability of ammonia. Alteration of highly conserved residues within the kinase/phosphatase domain of NRII revealed that the positive and negative regulatory functions of NRII could be genetically separated and that negative regulation by NRII did not require the highly conserved His-139, Glu-140, Asn-248, Asp-287, Gly-289, Gly-291, Gly-313, or Gly-315 residue. These mutations affected the positive regulatory function of NRII to various extents. Certain substitutions at codons 139 and 140 resulted in mutant NRII proteins that were transdominant negative regulators of glnA and the Ntr regulon even in the absence of nitrogen limitation. In addition, we examined three small deletions near the 3' end of the gene encoding NRII; these resulted in altered proteins that retained the negative regulatory function but were defective to various extents in the positive regulatory function. A truncated NRII protein missing the C-terminal 59 codons because of a nonsense mutation at codon 291 lacked entirely the positive regulatory function but was a negative regulator of glnA even in the absence of nitrogen limitation. Thus, we have identified both point and deletion mutations that convert NRII into a negative regulator of glnA and the Ntr regulon irrespective of the nitrogen status of the cell.     

Nucleotide sequence of the glnA-glnL intercistronic region of Escherichia coli

The nucleotide (nt) sequence of a 682-bp fragment containing the 3' end of the glnA gene, the region between the glnA and glnL genes, and the 5' end of the glnL gene from Escherichia coli was determined. This segment contains the region coding for the last 107 amino acids (aa) of glutamine synthetase, including the adenylylation site of this enzyme. The analysis of this sequence revealed two REP sequences, a Rho-independent terminator, the putative glnL promoter and the possible binding site for the glnG product, NRI.     

Mechanism of the PII-activated phosphatase activity of Escherichia coli NRII (NtrB): how the different domains of NRII collaborate to act as a phosphatase

The phosphatase activity of the homodimeric NRII protein of Escherichia coli is activated by the PII protein and requires all three domains of NRII. Mutations in the N-terminal domain (L16R), central domain (A129T), C-terminal domain PII-binding site (S227R), and C-terminal domain ATP-lid (Y302N) of NRII result in diminished phosphatase activity. Here, we used heterodimers formed in vitro from purified homodimeric proteins to study the phosphatase activity. A129T, S227R, and Y302N mutant subunits and A129T/S227R, A129T/Y302N, and S227R/Y302N double-mutant subunits formed stable heterodimers and were amenable to analysis; heterodimers containing these mutant subunits in various combinations were formed and their activities assessed. Complementation of the PII-activated phosphatase activity was observed in heterodimers containing S227R and Y302N subunits and in heterodimers containing A129T and Y302N subunits, but not in heterodimers containing A129T and S227R subunits. Complementation of the PII-activated phosphatase activity was also observed in heterodimers containing A129T/S227R and Y302N subunits, but not in heterodimers containing A129T/Y302N and S227R subunits. Finally, inclusion of an S227R/Y302N subunit in a heterodimer with a subunit having wild-type phosphatase activity resulted in a dramatic decrease in phosphatase activity, while inclusion of an A129T/S227R subunit did not. These results suggest that the phosphatase activity of NRII requires the collaboration of the PII-binding site from one subunit of the dimer, the central domain from the same subunit, and the ATP-lid from the opposing subunit, in addition to the undefined N-terminal domain requirement(s).     

Effects of glnL and other regulatory loci on regulation of transcription of glnA-lacZ fusions in Klebsiella aerogenes

Mutants of Klebsiella aerogenes containing genetic fusions of glnA to lacZ were isolated by using Mu dl (lac, bla) bacteriophage and a Mu Kmr helper phage with the host range of bacteriophage P1. Synthesis of beta-galactosidase in these strains is regulated in response to nitrogen metabolites and regulatory gln loci and is rendered constitutive by a mutation in the linked glnL gene. Complementation studies indicated that glnL is a separate locus from glnA and glnG and that insertions in glnA are partially polar on glnL expression. These results support the hypothesis that glnA, glnL, and glnG are organized in an operon with multiple promoters.     

Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII

Previous studies have established that the Escherichia coli protein kinase/phosphatase nitrogen regulator II (NRII also known as NtrB) becomes autophosphorylated on a histidine residue when incubated with ATP. We show that the major site at which NRII was autophosphorylated was contained within a peptide consisting of amino acid residues 136-142 of NRII, and thus probably corresponds to His-139. A minor site of phosphorylation, accounting for about 2% of the phosphate in NRII-P, was found in a peptide that corresponds to residues 158-169.