Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii.

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long-chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.

The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins.

The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented. The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da. The nifF product thus belongs to the long-chain class of flavodoxins. It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans. The long chain flavodoxins thus seem to constitute a well-conserved sub-group. The homology with the A. vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.

The nitrogen fixation genes of Klebsiella pneumoniae: a model system.

The genetics of nitrogen fixation and its regulation has been intensively studied in Klebsiella pneumoniae. The resulting model, whilst not complete, is proving very useful in understanding this process in other organisms.

Tumor-inducing (Ti) plasmids of Agrobacterium share extensive regions of DNA homology.

Labeled Ti plasmid DNAs from diverse Agrobacterium strains were hybridized to Southern blots of pTi-B6-806 plasmid DNA digest fragments of known map order. The map location of DNA sequences common to all Ti plasmids was found to be extensive, consistent with the view that Ti plasmids have evolved from a common ancestral plasmid.

Sequence of nifL from Klebsiella pneumoniae: mode of action and relationship to two families of regulatory proteins.

We present the nucleotide sequence of K. pneumoniae nifL, which negatively regulates nif transcription in response to oxygen and fixed nitrogen. It shows partial sequence homology to the general nitrogen regulatory proteins NtrB of K. pneumoniae and Bradyrhizobium parasponiae. This homology is weaker than that shown between the NifA and NtrC activator components of the nif and general nitrogen control systems. The N-terminal section of the NifL protein includes a structural duplication sharing sequence homology with part of NtrB, and a region containing a cysteine pair which might be implicated in redox control Unlike NtrB, NifL appears to lack a DNA-binding motif, consistent with evidence that NifL represses by interacting directly with NifA. The C-terminal section of NifL shows clear homology to NtrB and to a family of proteins involved in transcriptional control or chemotaxis, each of which probably interacts with a member of the family of regulatory proteins showing homology to NtrC.

The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins.

Evidence is presented that establishes a novel class of interdomain linkers, named Q-linkers, as a defined element of protein structure. Q-linkers occur at the boundaries of functionally distinct domains in a widespread set of bacterial regulatory and sensory transduction proteins, typified by the nitrogen regulatory proteins, NtrB, NtrC, NifA and NifL. Q-linkers are not strongly conserved in sequence in otherwise homologous proteins, are approximately 15-25 residues long and relatively rich in glutamine, arginine, glutamate, serine and proline, and are assigned as 'coil', with a very low probability of alpha or beta structure, by eight secondary structure prediction methods. Hydrophobic amino acids are spaced with a periodicity of approximately 4-5 residues in the C-terminal 15 residues of these sequences. A pattern discriminator is presented that incorporates these properties and is used to predict segments resembling Q-linkers in sequence databases. Insertions of four and eight amino acids, constructed in the Q-linker sequences of NtrC and NifA, were found to have no effect on the function of the proteins in signal transduction and transcriptional activation. However, when NtrC was expressed as two separate polypeptides, consisting of the domains normally joined by the Q-linker, the construct failed to function. These results suggest that the Q-linker serves a simple but essential role in tethering the structurally-distinct but interacting domains of the protein. Q-linkers are therefore potentially applicable as domain fusion junctions for engineered chimaeric multidomain proteins expressed in enteric bacterial systems.

The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae.

A model for the domain structure of sigma 54-dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of the N-terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over-expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of the N-terminal domain of NifA reduces activation 3-4-fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of the N-terminal domain is to block the action of NifL in derepressing conditions. The C-terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP-binding motif of the NtrC central domain, Ser169 to Ala, also abolished activator function. Exchanging the N-terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia coli OmpR, did not produce any hybrid activity, suggesting that N-terminal domains in the native proteins specifically recognize the rest of the molecule.

The boundaries and copy numbers of Ti plasmid T-DNA vary in crown gall tumors.

The Ti plasmid DNA maintained in octopine-type crown gall tumor lines is variable, but always includes at least part of the Ti plasmid that maps over the region of Hind III fragment 1 of pTiB6-806. The right-hand boundary of transferred DNA (T-DNA) varies considerably among the three independent tumor lines examined; the left boundary was not located definitively. The T-DNA of two sibling clones of the same tumor line, E1 and E9, appears identical. The copy number of T-DNA in E9 tumor DNA appears higher for the right end (about 30 copies) than for the left end (approximately 1 copy).

Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.