A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier to deciphering these sequences and understanding the relations among them is the difficulty of detecting subtle local residue patterns common to multiple sequences. Such patterns frequently reflect similar molecular structures and biological properties. A mathematical definition of this "local multiple alignment" problem suitable for full computer automation has been used to develop a new and sensitive algorithm, based on the statistical method of iterative sampling. This algorithm finds an optimized local alignment model for N sequences in N-linear time, requiring only seconds on current workstations, and allows the simultaneous detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix-turn-helix proteins, lipocalins, and prenyltransferases.

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Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment

Protein phosphorylation and regulation of adaptive responses in bacteria.

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.

PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light

Biological nitrogen fixation (BNF) is the process of the reduction of dinitrogen from the air to ammonia carried out by a large number of species of free-living and symbiotic microbes called diazotrophs. BNF presents an inexpensive and environmentally sound, sustainable approach to crop production and constitutes one of the most important Plant Growth Promotion (PGP) scenarios. Here I will summarize various aspects of BNF, including the dinitrogen reduction catalysed reaction carried out by “nitrogenase” and the enzymes/genes involved and their regulation, the inherent “oxygen paradox” , the identification of diazotrophs, sustainable agricultural uses of BNF, symbiotic plant-diazotroph interactions and endophytic diazotrophs, data from the field, and future prospects in BNF.

Biological Nitrogen Fixation

Short protocols in molecular biology

Nitrogen fixation (nif) genes in Klebsiella pneumoniae are controlled by a transcriptional activation mechanism which requires the ntrA gene product acting concert with either the ntrC or nifA products (reviewed in ref. 1). The nif promoters lack the canonical −35, −10 promoter elements and instead their activation requires nucleotide sequences located around −24 and −12 (refs 2–4). We have now identified and characterized a further essential promoter sequence in the K. pneumoniae nifH, nifU, nifB and ORF promoters, located more than 100 base pairs (bp) from the transcription start site. This promoter element is required for nifA- but not ntrC-mediated activation and for the inhibition of chromosomal nif expression observed when cells harbour multiple copies of certain nif promoters5. The upstream sequence is conserved among 10 Rhizobium and 2 Azotobacter nif promoters. We show that the positioning and orientation of the upstream sequence is not critical for promoter activity up to a distance of 2 kilobases (kb) and that the upstream sequence is itself transcriptionally inactive, probably acting in cis with the downstream sequences to produce a fully active promoter. Thus, an extended promoter structure is required for nif gene expression.

Upstream activator sequences are present in the promoters of nitrogen fixation genes

The nitrogen fixation (nif) genes of Klebsiella pneumoniae are specifically regulated by the products of the nifLA operon. We have located the promoter of this operon, and identified sequences required for nifLA transcription. Transcription from this promoter is shown to be positively regulated by the ntrC gene product (which coordinates the expression of many operons required for nitrogen assimilation) and also autogenously by the product of the nifA gene.

Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae.

Summary

A model for the domain structure of σ54-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, Ser 169 to Ala, also abolished activator function. Exchanging the N-terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia co/r OmpR, did not produce any hybrid activity, suggesting that N-terminal domains in the native proteins specifically recognize the rest of the molecule.

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

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C-terminal domain to the histidine protein kinases (HPKs) than is K. pneumoniae NifL. In particular A. vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems. Both NifL proteins are homologous in their N-termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio-opsin, the expression of which is oxygen sensitive. The same region showed homology to the haem-binding N-terminal domain of the Rhizobium meliloti fixL gene product, an oxygen-sensing protein. Like K. pneumoniae NifL, A. vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH+4 or oxygen. The sequences found homologous in the C-terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing. An in-frame deletion mutation in the nifL coding region resulted in loss of repression by NH+4 and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, nifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expression from the A. vinelandii promoter requires neither RpoN nor NtrC.

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii

The activity of a truncated form of Herbaspirillum seropedicae NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O2 sensitivity or Fe dependence. The model for nitrogen control involving PII could thus apply to the proteobacteria at large. NifA may have a role in controlling ADP-ribosylation of nitrogenase in Azospirillum brasilense.

Martin Drummond

Papers

Upstream activator sequences are present in the promoters of nitrogen fixation genes

Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae.

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

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii

Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen.