Domain Interactions on the ntr Signal Transduction Pathway: Two-Hybrid Analysis of Mutant and Truncated Derivatives of Histidine Kinase NtrB

We have used the yeast two-hybrid system to analyze protein-protein interactions mediated by domains of regulatory proteins of the ntr signal transduction system, including interactions among NtrB derivatives and their interactions with NtrC and PII from Klebsiella pneumoniae. Interactions took place only between proteins or protein domains belonging to the ntr signal transduction system and not between proteins or domains from noncognate regulators. NtrB and its transmitter domain, but not NtrC, CheA, or the cytoplasmic C terminus of EnvZ, interacted with PII. In addition, interaction of NtrB with NtrC, but not with PII, depended on the histidine phosphotransfer domain. Point mutation A129T, diminishing the NtrC phosphatase activity of NtrB, affected the strength of the signals between NtrC and the transmitter module of NtrB but had no impact on PII signals, suggesting that A129T prevents the conformational change needed by NtrB to function as a phosphatase for NtrC, rather than disturbing binding to PII.     

Structural analysis of the ntrBC genes of deep-sea piezophilic Shewanella violacea

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.     

Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression

Abstract The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli . The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris . Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NR_II (NtrB) and NR_I (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NR_I. NR_II was not detectable using the methods employed.     

Mechanism of regulation of the bifunctional histidine kinase NtrB in Escherichia coli

NtrB is the bifunctional histidine kinase for nitrogen regulation. Dependent on the availability of nitrogen, it either autophosphorylates and serves as the phosphodonor for its cognate response regulator, NtrC, or, it promotes the rapid dephosphorylation of NtrC-P. The activity of NtrB depends on the interaction of two subdomains within its transmitter domain, the H-domain and the kinase domain. Both phosphotransfer activity and phosphatase activity reside in the H-domain. When separately expressed, this domain acts as a phosphatase. Interaction with the kinase domain results in the inhibition of the phosphatase activity and the phosphorylation of the conserved histidine of the H-domain.     

Deletion analysis of the nitrogen fixation regulatory gene,nifL of Klebsiella pneumoniae

A number of in-frame deletions have been constructed in the Klebsiella pneumoniae regulatory gene nifL. The effects of each nifL mutation on NifA-mediated expression from the nifH promoter of K. pneumoniae have then been assessed with respect to both nitrogen and oxygen control. These experiments indicate that, in contrast to the situation with the homologous regulatory proteins NtrB and NtrC, NifA activity is not impaired in the absence of NifL. We conclude that the only function of NifL is to inactivate NifA in response to an increase in the nitrogen or oxygen status of the cell.     

I do it my way: regulation of ammonium uptake and ammonium assimilation in Corynebacterium glutamicum

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, recent advances in our knowledge of ammonium uptake, its assimilation, and connected regulatory systems in Corynebacterium glutamicum are discussed with respect to the situation in the bacterial model organisms Escherichia coli and Bacillus subtilis. The regulatory network of nitrogen control in C. glutamicum differs substantially from that in these bacteria, for example, by the presence of AmtR, the unique "master regulator" of nitrogen control, the absence of a NtrB/NtrC two-component signal transduction system, and a different sensing mechanism in C. glutamicum.