Iron homeostasis is vital for many cellular processes and requires a precise regulation. Several iron efficient plants respond to iron starvation with the excretion of riboflavin and other flavins. Basic helix–loop–helix transcription factors (TF) are involved in the regulation of many developmental processes, including iron assimilation. Here we describe the isolation and characterisation of two Arabidopsis bHLH TF genes, which are strongly induced under iron starvation. Their heterologous ectopic expression causes constitutive, iron starvation independent excretion of riboflavin. The results show that both bHLH TFs represent an essential component of the regulatory pathway connecting iron deficiency perception and riboflavin excretion and might act as integrators of various stress reactions.

The Corynebacterium glutamicum glutamine synthetase I (GSI) structural gene glnA was cloned by a PCR approach using oligonucleotide primers derived from conserved amino acid sequences of the GSI proteins from various bacteria. Disruption or deletion of this gene in C. glutamicum led to a glutamine auxotrophic phenotype and complete loss of glutamine synthetase activity, indicating the key role of this enzyme in nitrogen metabolism. Additionally, indications for a second glutamine synthetase, GSII, were found.

Iron mobilization responses are induced by low iron supply at transcriptional level. In tomato, the basic helix-loop-helix gene FER is required for induction of iron mobilization. Using molecular-genetic techniques, we analyzed the function of BHLH029, named FRU (FER-like regulator of iron uptake), the Arabidopsis thaliana homolog of the tomato FER gene. The FRU gene was mainly expressed in roots in a cell-specific pattern and induced by iron deficiency. FRU mutant plants were chlorotic, and the FRU gene was found necessary for induction of the essential iron mobilization genes FRO2 (ferric chelate reductase gene) and IRT1 (iron-regulated transporter gene). Overexpression of FRU resulted in an increase of iron mobilization responses at low iron supply. Thus, the FRU gene is a mediator in induction of iron mobilization responses in Arabidopsis, indicating that regulation of iron uptake is conserved in dicot species.

In Corynebacterium glutamicum, a Gram-positive soil bacterium widely used in the industrial production of amino acids, two genes encoding (putative) ammonium uptake carriers have been described. The isolation of amt was the first report of the sequence of a gene encoding a bacterial ammonium uptake system combined with the characterization of the corresponding protein. Recently, a second amt gene, amtB, with so far unknown function, was isolated. The isolation of this gene and the suggestion of a new concept for ammonium acquisition prompted the reinvestigation of ammonium transport in C. glutamicum. In this study it is shown that Amt mediates uptake of (methyl)ammonium into the cell with high affinity and strictly depending on the membrane potential. As shown by the determination of K:(m) at different pH values, ammonium/methylammonium, but not ammonia/methylamine, are substrates of Amt. AmtB exclusively accepts ammonium as a transport substrate. In addition, hints of another, until now unknown, low-affinity, ammonium-specific uptake system were found.

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram‐negative enterobacterium Escherichia coli, the NtrB/C two‐component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram‐positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and β‐galactosidase activity in reporter strains. The repressor‐encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR‐expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1−4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one‐hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB–glnK–glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl‐removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram‐positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram‐positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram‐negative enterobacteria.

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PII (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.

The construction of new Corynebacterium glutamicum/Escherichia coli shuttle vectors with improved cloning properties and an increased chloramphenicol resistance (50 μg ml−1 MIC) is described. A modified glnB gene encoding a Strep-tag II domain modified PII protein was expressed in C. glutamicum and streptavidin affinity chromatography was used to purify this protein.