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

Genome sequence of the bioplastic-producing ‘‘Knallgas’’ bacterium Ralstonia eutropha H16

The H2-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H2 and CO2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism’s ability to produce and store large amounts of poly[R-(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility

Carbonic Anhydrase Is Essential for Growth of Ralstonia eutropha at Ambient CO(2) Concentrations

Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO(2)-requiring (HCR) phenotype. Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO(2). In the present study, a gene designated can and encoding a β-carbonic anhydrase (CA) was identified as the site altered in strain 25-1. The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can. A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1. Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA. The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO(4) for maximal activity. Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO(2) concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO(2) concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified

Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria

Structural and regulatory genes encoding enzymes and proteins of the reductive pentose phosphate pathway have been isolated from a number of bacteria recently. In the phototroph Rhodobacter sphaeroides, and in two chemoautotrophic bacteria, Alcaligenes eutrophus and Xanthobacter flavus, these genes have been found in distinct operons. However, in these three organisms and in other bacteria where certain of these genes have been discovered, a uniform nomenclature to designate these genes has been lacking. This report represents an effort to provide uniformity to the designation of these genes from all bacteria.

Ralstonia eutropha H16 Flagellation Changes According to Nutrient Supply and State of Poly(3-Hydroxybutyrate) Accumulation▿

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella