The manganese and calcium ions of photosynthetic oxygen evolution.

Some bacteria have the remarkable capacity to fix atmospheric nitrogen to ammonia under ambient conditions, a reaction only mimicked on an industrial scale by a chemical process that requires high temperatures, elevated pressure and special catalysts. The ability of microorganisms to use nitrogen gas as the sole nitrogen source and engage in symbioses with host plants confers many ecological advantages, but also incurs physiological penalties because the process is oxygen sensitive and energy dependent. Consequently, biological nitrogen fixation is highly regulated at the transcriptional level by sophisticated regulatory networks that respond to multiple environmental cues.

Genetic regulation of biological nitrogen fixation

Two-component signal transduction systems enable bacteria to sense, respond, and adapt to a wide range of environments, stressors, and growth conditions. In the prototypical two-component system, a sensor histidine kinase catalyzes its autophosphorylation and then subsequently transfers the phosphoryl group to a response regulator, which can then effect changes in cellular physiology, often by regulating gene expression. The utility of these signaling systems is underscored by their prevalence throughout the bacterial kingdom and by the fact that many bacteria contain dozens, or sometimes hundreds, of these signaling proteins. The presence of so many highly related signaling proteins in individual cells creates both an opportunity and a challenge. Do cells take advantage of the similarity between signaling proteins to integrate signals or diversify responses, and thereby enhance their ability to process information? Conversely, how do cells prevent unwanted cross-talk and maintain the insulation of distinct pathways? Here we address both questions by reviewing the cellular and molecular mechanisms that dictate the specificity of two-component signaling pathways.

Specificity in Two-Component Signal Transduction Pathways

Nitrogen control in bacteria.

Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation) Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase

Copper is an essential cofactor of various enzymes, but free copper is highly toxic to living cells. To maintain cellular metabolism at different ambient copper concentrations, bacteria have evolved specific copper homeostasis systems that mostly act as defence mechanisms. As well as under free-living conditions, copper defence is critical for virulence in pathogenic bacteria. Most bacteria synthesize P-type copper export ATPases as principal defence determinants when copper concentrations exceed favourable levels. In addition, many bacteria utilize resistance-nodulation-cell division (RND)-type efflux systems and multicopper oxidases to cope with excess copper. This review summarizes our current knowledge on copper-sensing transcriptional regulators, which we assign to nine different classes. Widespread one-component regulators are CueR, CopY and CsoR, which were initially identified in Escherichia coli, Enterococcus hirae and Mycobacterium tuberculosis, respectively. CueR activates homeostasis gene expression at elevated copper concentrations, while CopY and CsoR repress their target genes under copper-limiting conditions. Besides these one-component systems, which sense the cytoplasmic copper status, many Gram-negative bacteria utilize two-component systems, which sense periplasmic copper concentrations. In addition to these well-studied transcriptional factors, copper control mechanisms acting at the post-transcriptional and the post-translational levels will be discussed.

Copper-responsive gene regulation in bacteria

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.

Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus.

In most bacteria, nitrogen metabolism is tightly regulated and PII proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK) encoding PII-like proteins. The glnB gene forms part of a glnB–glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both PII-like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB–glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB–glnK double mutant. Furthermore, the PII-like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB–glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.

Bernd Masepohl

Papers

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase

Copper-responsive gene regulation in bacteria

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus.

Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus.