Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different subunits. In addition to the homologues of these subunits, the mitochondrial complex contains approximately 31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli mutants, in which the nuo-genes coding the subunits of complex I were individually disrupted by an insertion of a resistance cartridge to determine whether they are required for the assembly of a functional complex I. No complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron-sulfur cluster N2 was detected in one mutant.

The response of Mannheimia haemolytica to iron limitation: implications for the acquisition of iron in the bovine lung.

Mannheimia haemolytica is the major causative agent of shipping fever, a severe pneumonia in cattle causing high morbidity and mortality. A prerequisite of successful lung colonization by M. haemolytica is the necessity to adapt to the paucity of iron. The lack of genome information has precluded an assessment of the genetic repertoire available to M. haemolytica to adapt to low iron environments. To close this knowledge-gap, we have determined 90% of a virulent M. haemolytica serotype A1 genome sequence and produced a microarray in order to study gene expression under iron-limiting growth for 15, 30 and 60 min. M. haemolytica responded to iron limitation by the up-regulation of transcripts coding for receptors and ABC-type transporters of transferrin, haemoglobin, haem and siderophores. Real time PCR analysis of lung tissue from Mannheimia-infected calves demonstrated the in vivo transcription of two potential haemoglobin receptors, hmbR1 and hmbR2. The relative hmbR1 and hmbR2 transcript levels in the infected lung tissue were comparable to the induced levels observed under iron-limiting growth, demonstrating in vivo induction of receptor transcription in the context of an infection. When the iron response of M. haemolytica was compared to the iron response of Pasteurella multocida, another pathogen colonizing the bovine lung, only few homologous genes were induced in both organisms. These included the haemoglobin receptor hmbR2 and the periplasmic transport systems yfeABCD and fbpABC. The comparative analysis suggests that the two pathogens use different strategies to adapt to the iron-limiting environment in the bovine host.

Large-scale production of the immunomodulator c-di-GMP from GMP and ATP by an enzymatic cascade.

(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1ß, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

Iron-sulfur cluster N2 of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is located on subunit NuoB.

The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction. There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping. However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate. Complex I from Escherichia coli consists of 13 polypeptides called NuoA to N. Either subunit NuoB or NuoI could harbor Fe/S cluster N2. Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters. Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex. Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype. Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A. N2 was also detected in the complex isolated from the mutant NuoI C102A. From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.