Extending the models for iron and sulfur oxidation in the extreme Acidophile Acidithiobacillus ferrooxidans

<p>Abstract</p> <p>Background</p> <p><it>Acidithiobacillus ferrooxidans </it>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 <it>A. ferrooxidans </it>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 <it>A. ferrooxidans </it>in order to comprehend the full metabolic potential of the pangenome of the genus.</p> <p>Results</p> <p>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 <it>A. ferrooxidans </it>ATCC23270. These include for iron oxidation: <it>cup </it>(copper oxidase-like), <it>ctaABT </it>(heme biogenesis and insertion), <it>nuoI </it>and <it>nuoK </it>(NADH complex subunits), <it>sdrA1 </it>(a NADH complex accessory protein) and <it>atpB </it>and <it>atpE </it>(ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (<it>rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB</it>) encoding three sulfurtransferases and a heterodisulfide reductase complex, <it>sat </it>potentially encoding an ATP sulfurylase and <it>sdrA2 </it>(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 (<it>ctaRUS</it>) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome <it>aa</it>₃oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool.</p> <p>Conclusion</p> <p>Bioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in <it>A. ferrooxidans </it>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.</p

Novel Inducers of the Envelope Stress Response BaeSR in Salmonella Typhimurium: BaeR Is Critically Required for Tungstate Waste Disposal

The RpoE and CpxR regulated envelope stress responses are extremely important for SalmonellaTyphimurium to cause infection in a range of hosts. Until now the role for BaeSR in both the Salmonella Typhimurium response to stress and its contribution to infection have not been fully elucidated. Here we demonstrate stationary phase growth, iron and sodium tungstate as novel inducers of the BaeRregulon, with BaeR critically required for Salmonella resistance to sodium tungstate. We show that functional overlap between the resistance nodulation-cell division (RND) multidrug transporters, MdtA, AcrD and AcrB exists for the waste disposal of tungstate from the cell. We also point to a role for enterobactinsiderophores in the protection of enteric organisms from tungstate, akin to the scenario in nitrogen fixing bacteria. Surprisingly, BaeR is the first envelope stress response pathway investigated in S. Typhimurium that is not required for murine typhoid in either ityS or ityR mouse backgrounds. BaeR is therefore either required for survival in larger mammals such as pigs or calves, an avian host such as chickens, or survival out with the host altogether where Salmonella and related enterics must survive in soil and water

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B

Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium

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

Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism

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

Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation

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 c1, cytochrome b and a Rieske protein, that constitute a bc1, 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 c4 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 contai

Structural basis for the oxidation of thiosulfate by a sulfur cycle enzyme

Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains. This metabolism is a key component of the biogeochemical sulfur cycle. The SoxAX protein is a heterodimeric c-type cytochrome involved in thiosulfate oxidation. The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 Ã… resolution in the oxidized state and at 1.5 Ã… resolution in the dithionite-reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate. The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination. This unusual post-translational modification is also seen in sulfurtransferases such as rhodanese. Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential. These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation. The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33