Genes and pathways for CO2 fixation in the obligate, chemolithoautotrophic acidophile, Acidithiobacillus ferrooxidans, Carbon fixation in A. ferrooxidans

<p>Abstract</p> <p>Background</p> <p><it>Acidithiobacillus ferrooxidans </it>is chemolithoautotrophic γ-proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO₂uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study.</p> <p>Results</p> <p>Four gene clusters (termed <it>cbb1-4</it>) in the <it>A. ferrooxidans </it>genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson-Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO₂-concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon <it>cbb1 </it>is divergently transcribed from a gene, <it>cbbR</it>, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma⁷⁰-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N₁₁-A/TNA-N₇TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the <it>cbb1</it>, <it>cbb2 </it>and <it>cbb3 </it>operons, demonstrating that the predicted CbbR-binding sites are functional <it>in vitro</it>. However, CbbR failed to bind the upstream region of the <it>cbb4 </it>operon that contains <it>cbbP</it>, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The <it>cbb3 </it>operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO₂fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of <it>A. ferrooxidans </it>to fix CO₂at different concentrations of CO₂.</p> <p>Conclusions</p> <p><it>A. ferrooxidans </it>has features of <it>cbb </it>gene organization for CO₂-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the <it>cbbP </it>gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation.</p

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

Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans

This study was motivated by surprising gaps in the current knowledge of microbial inorganic carbon (Ci) uptake and assimilation at acidic pH values (pH &lt; 3). Particularly striking is the limited understanding of the differences between Ci uptake mechanisms in acidic versus circumneutral environments where the Ci predominantly occurs either as a dissolved gas (CO2) or as bicarbonate (HCO3-), respectively. In order to gain initial traction on the problem, the relative abundance of transcripts encoding proteins involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using ferrous iron as an energy source, although they are able to grow at pH 5 when using sulfur as an energy source. The relative abundance of transcripts of five operons (cbb1-5) and one gene cluster (can-sulP) was monitored by RT-qPCR and, in selected cases, at the protein level by Western blotting, when cells were grown under different regimens of CO2 concentration in elemental sulfur. Of particular note was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP, previously annotated as a sulfate transporter, is a novel type of bicarbonate transporter. A conceptual model of CO2 fixation was constructed from combined bioinformatic and experimental approaches that suggests strategies for providing ecological flexibility under changing concentrations of CO2 and provides a portal to elucidating Ci uptake and regulation in acidic conditions. The results could advance the understanding of industrial bioleaching processes to recover metals such as copper at acidic pH. In addition, they may also shed light on how chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally occurring low pH environments

Organization of the 16s‐23s intergenic spacer region of the two rRNA operons from thiobacillus ferrooxidans

The recombinant plasmid pTR‐3 was previously shown to contain part of the 16S and 23S ribosomal RNA genes and the spacer region between the two genes from operon rrnT, from Thiobacillus ferrooxidans. The spacer region was subjected to deletions, using the exonuclease III nested deletions procedure, and the resulting fragments were sequenced. tRNAile‐ and tRNAala‐like sequences were identified near the 3’ end of16S rRNA gene. The spacer DNAs from both rRNA operons were amplified by the polymerase chain reaction (PCR) and the sequences were compared. No differences were observed. A DNA segment identical to putative box A of the antiterminator sequence of Mycoplasma sp. was identified. Comparison between the sequence of the spacer region from strains Torma and A4 showed some minor differences. It implies that only in strain A4 is a recognition site for the Avail restriction enzyme present. © 1992 Taylor & Francis Group, LLC

Notes &amp; Tips 3 An artifact in studies of gene regulation using b-galactosidase reporter gene assays

To explain this up-regulation, it is hypothesized that the 76 a-peptide of b-gal encoded by pUC18 is able to increase the activ-77 ity of the wild-type b-gal expressed from the intact chromosomal 78 copy of lacZ of E. coli H2331 Pfur To test this hypothesis, several different plasmids encoding the 80 a-peptide of b-gal were transformed into the host E. coli BL21(DE3) 81 that contains lacZ expressed from its native promoter (P LAC

SHORT COMMUNICATION - An RT-PCR artifact in the characterization of bacterial operons

During the course of experiments involving RT-PCR to investigate the co-transcription of the pet and res operons of the bacterium Acidithiobacillus ferrooxidans we observed that the mRNA of certain gene pairs can be amplified by RT-PCR in the absence of added RT-primer. We provide experimental evidence that eliminates, as explanations for the observed results, the possible global contamination of the RNA preparation by genomic DNA or by contaminating single stranded DNA or RNA fragments. We suggest that the results can be explained by self-priming of the RNA perhaps as a result of secondary structure configurations that provide a suitable 3’-terminus to prime the reverse transcriptase. This type of artifact can be recognized by carrying out a control in which exogenously added RT-primers are left out of the reaction. Such a control is essential if RT-PCR is to be used to investigate and correctly identify operons. RT-PCR is also an important tool for the amplification of eukaryotic mRNAs and our observations may also be applicable to these systems

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

The genome of the acidophilic, proteobacterium Acidithiobacillus ferrooxidans, contains linked but divergently oriented genes, termed afeI and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afeI. A cloned copy of afeI, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C14. This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur- rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts o

Properties of a cyclic nucleotide phosphodiesterase of amphibian oocytes that is activated by calmodulin and calcium, by tryptic proteolysis, and by phospholipids

A calmodulin-Ca2+-stimulated cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which hydrolyzed both cGMP and cAMP has been purified about 2000-fold from ovaries of the amphibian Xenopus laevis. Gel filtration through Sephadex G-200 indicated a molecular weight of 140,000. A single, major protein band of molecular weight 66,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the stimulation by calmodulin-Ca2+, the enzyme was activated 5- to 10-fold by proteolysis and by certain phospholipids. Trypsin activation of the enzyme caused a reduction in the native molecular weight to 90,000 and a loss of the capacity to be stimulated by calmodulin-Ca2+ or by phospholipids. The phosphodiesterase was stimulated by low concentrations (0.1 μg/ml) of lysophosphatidylcholine and lysophosphatidylethanolamine. This response did not require calcium ions. Phosphatidylinositol, fatty acids, progesterone, and phospholipase C had little or no effect on activity. Simultaneous addition of 1 mM 2-chloro-lo-(3-aminopropyl)phenothiazine and lysophosphatidylcholine to the enzyme did not diminish the stimulatory effect of the phospholipid. The activation of the enzyme by all three agents resulted in an increase in the maximum velocity of the reaction without significant modification of the apparent Km values for cGMP (5 PM) or CAMP (30 PM). It was suggested that trypsin removed an inhibitory domain from the enzyme and that calmodulin and phospholipids interact with this same domain, eliminating its capacity to inhibit the active center of the enzym