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Abstract Background Acidithiobacillus 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. Results Bioinformatic-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. Conclusion Bioinformatic 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.

Toll-like receptor 4 (TLR4) senses bacterial LPS and is required for the control of systemic Salmonella enterica serovar Typhimurium infection in mice. The mechanisms of TLR4 activation and its downstream signalling cascades are well described, yet the direct effects on the pathogen of signalling via this receptor remain unknown. To investigate this we used microarray-based transcriptome profiling of intracellular S. Typhimurium during infection of primary bone marrow-derived macrophages from wild-type and TLR4-deficient mice. We identified 1 7 S. Typhimurium genes that were upregulated in the presence of functional TLR4. Nine of these genes have putative functions in oxidative stress resistance. We therefore examined S. Typhimurium gene expression during infection of NADPH oxidase-deficient macrophages, which lack normal oxidative killing mechanisms. We identified significant overlap between the 'TLR4-responsive' and 'NADPH oxidase-responsive' genes. This is new evidence for a link between TLR4 signalling and NADPH oxidase activity. Interestingly, with the exception of a dps mutant, S. Typhimurium strains lacking individual TLR4- and/or oxidative stress-responsive genes were not attenuated during intravenous murine infections. Our study shows that TLR4 activity, either directly or indirectly, induces the expression of multiple stress resistance genes during the intracellular life of S. Typhimurium.

Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petllABC gene cluster that encodes the three proteins, cytochrome [c.sub.1], cytochrome b and a Rieske protein, that constitute a [bc.sub.1] electron-transfer complex. RT-PCR and Northern blotting show that the petllABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the [c.sub.4] family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron-sulfur protein, suggesting that the six genes constitute an operon, termed the petll operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petl operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petl is transcribed mainly in cells grown in medium containing iron, whereas petll is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petl and petll operons. A model is presented in which petl is proposed to encode the [bc.sub.1] complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petll is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional [bc.sub.1] complexes.

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.

Research has been conducted on the thiosulfate-induced cytochrome. The purification of this cytochrome has been performed and the results demonstrate that it is a heterodimeric protein that contains covalently bound heme groups.

Results point out that RecA protein has less influence in the DNA repair in Thiobacillus ferrooxidans as revealed by less sensitivity of recA mutants to the DNA-dmaging agents.

Research conducted on the gene encoding of the electron transfer proteins is discussed. The results demonstrate that the genes encoding four cytochrome c oxidase subunits belong to the same operon.

Global agricultural emissions of the greenhouse gas nitrous oxide (N2O) 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 (N2) via N2O 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 N2O to N2, is regulated in response to the extracellular copper concentration. We show that elevated levels of N2O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B12-dependent to vitamin B12-independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B12 riboswitches. This inhibitory effect of N2O can be rescued by addition of exogenous vitamin B12. [ABSTRACT FROM AUTHOR]

Studies the sequences of the cox genes encoding the subunits of an oxidase in the cells of Thiobacillus ferrooxidans (T. ferrooxidans). Northern and reverse transcription-polymerase chain reaction (PCR) analyses; Identification of transcriptional start sites; Redox proteins from the contranscription of genes involved in an electron transfer chain.

Global agricultural emissions of the greenhouse gas nitrous oxide (N2O) 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 (N2) via N2O 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 N2O to N2, is regulated in response to the extracellular copper concentration. We show that elevated levels of N2O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B12-dependent to vitamin B12-independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B12 riboswitches. This inhibitory effect of N2O can be rescued by addition of exogenous vitamin B12.

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.