BackgroundAcidithiobacillus ferrooxidans gains energy from the oxidation of ferrous iron and various reduced inorganic sulfur compounds at very acidic pH. Although an initial model for the electron pathways involved in iron oxidation has been developed, much less is known about the sulfur oxidation in this microorganism. In addition, what has been reported for both iron and sulfur oxidation has been derived from different A. ferrooxidans strains, some of which have not been phylogenetically characterized and some have been shown to be mixed cultures. It is necessary to provide models of iron and sulfur oxidation pathways within one strain of A. ferrooxidans in order to comprehend the full metabolic potential of the pangenome of the genus.ResultsBioinformatic-based metabolic reconstruction supported by microarray transcript profiling and quantitative RT-PCR analysis predicts the involvement of a number of novel genes involved in iron and sulfur oxidation in A. ferrooxidans ATCC23270. These include for iron oxidation: cup (copper oxidase-like), ctaABT (heme biogenesis and insertion), nuoI and nuoK (NADH complex subunits), sdrA1 (a NADH complex accessory protein) and atpB and atpE (ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB) encoding three sulfurtransferases and a heterodisulfide reductase complex, sat potentially encoding an ATP sulfurylase and sdrA2 (an accessory NADH complex subunit). Two different regulatory components are predicted to be involved in the regulation of alternate electron transfer pathways: 1) a gene cluster (ctaRUS) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome aa3 oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool.ConclusionBioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in A. ferrooxidans and suggests mechanisms for their regulation. The models provide unified and coherent descriptions of these processes within the type strain, eliminating previous ambiguity caused by models built from analyses of multiple and divergent strains of this microorganism.

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome c has been purified from the photosynthetic ␣-proteobacterium Rhodovulum sulfidophilum. This cytochrome c 551 is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome c 551 structural genes are part of an 11-gene sox locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome c 551 is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type R. sulfidophilum exhibit thiosulfate: cytochrome c oxidoreductase activity. However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c 551 . Gene clusters similar to the R. sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

Global agricultural emissions of the greenhouse gas nitrous oxide (N 2 O) have increased by around 20% over the last 100 y, but regulation of these emissions and their impact on bacterial cellular metabolism are poorly understood. Denitrifying bacteria convert nitrate in soils to inert di-nitrogen gas (N 2 ) via N 2 O and the biochemistry of this process has been studied extensively in Paracoccus denitrificans. Here we demonstrate that expression of the gene encoding the nitrous oxide reductase (NosZ), which converts N 2 O to N 2 , is regulated in response to the extracellular copper concentration. We show that elevated levels of N 2 O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B 12 -dependent to vitamin B 12 -independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B 12 riboswitches. This inhibitory effect of N 2 O can be rescued by addition of exogenous vitamin B 12 .denitrification | transcription | NosR | NosC G lobal atmospheric loading of the ozone-depleting greenhouse gas, nitrous oxide (N 2 O), is on the increase (1). Molecule for molecule, its radiative potential is ∼300-fold higher than carbon dioxide (2, 3), comprising ∼9% of global radiative forcing by greenhouse gases (4). In addition, atmospheric N 2 O is stable for ∼120 y. Approximately 70% of anthropogenic N 2 O loading arises from agriculture, mainly from the use of nitrogencontaining fertilizers by soil microbes for dissimilatory purposes. Taken together, these features make N 2 O an important target for mitigation strategies (5).N 2 O is an intermediate in the sequential reduction of nitrate (NO 3 − ) to di-nitrogen (N 2 ), via nitrite (NO 2 − ), nitric oxide (NO), and N 2 O, a process known as denitrification (6). Under certain conditions, the final step in denitrification is dispensed with and N 2 O is released into the atmosphere. One limiting factor in this process is copper (Cu) availability, the metal cofactor required by the N 2 O reductase (NosZ) that destroys N 2 O (5, 7, 8). During Cu-limitation the catalytic capacity of the Nos system may be exceeded by the rate of the preceding reactions that generate N 2 O (i.e., NO 3 − , NO 2 − , and NO reduction) and thus, N 2 O is emitted by denitrifying bacteria (7, 9, 10).Much attention has been given to the cytotoxic properties of NO as a free-radical and oxidant, but N 2 O is often described as a relatively inert intermediate of the nitrogen cycle. However, N 2 O exhibits cytotoxicity, as it is known to bind to and inactivate vitamin B 12 (B 12 ), an essential cellular cofactor in B 12 -dependent enzymes involved in methionine and DNA synthesis (11,12). B 12 also acts as a ligand for B 12 riboswitches that modulate gene expression in the absence of this cofactor (13,14). The possible impact of environmental N 2 O emissions on B 12 metabolism in microbiological communities has largely been ignored. As levels of N 2 O increase in the environment, there is a compelling argument for ...

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea (Pisum sativum) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants (nodC and nifH), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective (nifH) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo-inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a g-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch (Vicia hirsuta), producing similar results. In addition, vetch exhibited a local increase of nod gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere.

The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.

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The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N₂O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N₂O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N₂O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N₂O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N₂O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N₂O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N₂O production, and this can account for up to 20% of the nitrate catabolized.