Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

http://www.envismadrasuniv.org/pdf/microbial%20genome%20environment.pdf

Community structure and metabolism through reconstruction of microbial genomes from the environment

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

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Cytochrome bd is a respiratory oxidase in Escherichia coli and many other bacteria. It contains cytochromes b558, b595 and d as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases. The apparent affinities (Km) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome bo' were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e. oxygen) inhibition. Km values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and Ki values between 0.5 and 1.8 microM were obtained. Low temperature photodissociation of anoxic, CO-ligated membranes confirmed the absence of cytochrome bo' and revealed a high-spin b-type cytochrome identified as cytochrome b595 of the cytochrome bd complex. Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome b595 at a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome d. The results confirm that both high-spin haems in the cytochrome bd complex bind CO and demonstrate that oxygen can also react with both haems. Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome bd- and bo'-terminated respiratory pathways.

The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the structural gene operon, which is autogenously regulated.

Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions

The enzyme complex nitrogenase, which reduces N2 to NH+4, involves two redox proteins, both irreversibly damaged by O2 (ref. 1). Enzyme activity therefore requires anaerobic conditions, a source of reductant and a large amount of ATP (approximately 16 ATPs per N2). In both aerobic and facultative anaerobic N2-fixing bacteria, nitrogenase synthesis is regulated by O2 and NH+4, but in the aerobes there are also processes to protect the enzyme from O2 damage. The mechanisms of repression by O2 and NH+4 seem to be independent in the organisms so far examined. In the facultative anaerobe, Klebsiella pneumoniae, O2 was shown to repress nitrogenase synthesis in an NH+4-constitutive strain. The fusion of the Escherichia coli lacZ gene into each transcriptional unit of the nitrogen fixation (nif) gene cluster in K. pneumoniae has facilitated studies with O2, because expression from the various nif promoters results in an O2-stable product (beta-galactosidase). Notably, the nifHDK operon (the nitrogenase structural genes) was more sensitive to O2 repression than the nifLA operon (regulatory genes). The characterization of mutants, reported here, indicates the involvement of a nif-regulatory gene product in the mechanism of O2 control of nitrogenase synthesis.

Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae.

Certain mutations in the nifL gene of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster resulted in altered nif regulaiton such that nitrogenase synthesis was no longer repressed by low levels of exogenous fixed nitrogen, by oxygen or by high temperature. Introduction of a plasmid with a nifL+ allele restored fixed nitrogen and oxygen repression. We therefore conclude that the nifL product acts as a nif-specific repressor in response to these effectors.

Susan Hill

Papers

The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions

Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae.

Repressor properties of the nifL gene product in Klebsiella pneumoniae