No abstract

The nutritional versatility of facultative autotrophs requires efficient overall control of their metabolism. Most of these organisms are Proteobacteria that assimilate CO(2) via the highly energy-demanding Calvin-Benson-Bassham reductive pentose-phosphate cycle. The enzymes of the cycle are encoded by cbb genes organized in cbb operons differing in size and composition, although conserved features are apparent. Transcription of the operons, which may form regulons, is strictly controlled, being induced during autotrophic but repressed to varying extents during heterotrophic growth of the bacteria. The chemoautotroph Ralstonia eutropha is one of the organisms studied extensively for the mechanisms involved in the expression of cbb gene systems. CbbR is a LysR-type transcriptional regulator and the key activator protein of cbb operons. The cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. The activating function of CbbR seems to be modulated by metabolites signaling the nutritional state of the cell to the cbb system. Phosphoenolpyruvate is such a signal metabolite acting as a negative effector of R. eutropha CbbR, whereas NADPH has been proposed to be a coactivator of the protein in two other chemoautotrophs, Xanthobacter flavus and Hydrogenophilus thermoluteolus. There is evidence for the participation of additional regulators in cbb control. In the photoautotrophs Rhodobacter capsulatus and Rhodobacter sphaeroides, response regulator RegA of the global two-component signal transduction system RegBA serves this function. It is conceivable that specific variants of cbb control systems have evolved to ensure their optimal integration into regulatory networks operating in the diverse autotrophs characterized by different metabolic capabilities.

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 ␤-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.Carbon dioxide and bicarbonate (dissolved inorganic carbon [DIC]) are essential growth factors for bacteria. The metabolic need for DIC is evident in autotrophs utilizing CO 2 as the sole carbon source, but heterotrophs also fix significant amounts of both carbon species. Although sufficient CO 2 is produced during catabolism, deprivation of atmospheric CO 2 leads to growth inhibition or even death of heterotrophs (7,14,22). Pathogenic bacteria seem to be adapted to high DIC concentrations in their host environment, as they usually require 5 to 10% (vol/vol) CO 2 for growth (9,45,60). Furthermore, elevated DIC was found to shorten the lag phase and accelerate growth of bacteria even though the organisms were not generally dependent on high DIC concentrations (46,47,60). This "sparking effect" is most pronounced when cultures are inoculated at low cell densities. The need for DIC is generally attributed to CO 2 fixation in anaplerotic or other biosynthetic reactions. Consequently, the requirement is often satisfied by supplementation of the growth media with metabolites, particularly intermediates of the tricarboxylic acid cycle such as oxaloacetate and 2-oxoglutarate (28). Most high-CO 2 -requiring (HCR) mutants of Escherichia coli and other microorganisms regained the ability to grow at air concentrations of CO 2 (0.035% [vol/vol]) upon provision with appropriate metabolites, but some depended strictly on high CO 2 concentrations (5 to 10% [vol/vol]) (1,10,64). In contrast to their general DIC requirement, many microorganisms are inhibited by very high CO 2 concentrations (ca. 20% [vol/vol] and above), an ef...

The Calvin-Benson-Bassham cycle constitutes the principal route of CO2 assimilation in aerobic chemoautotrophic and in anaerobic phototrophic purple bacteria. Most of the enzymes of the cycle are found to be encoded by cbb genes. Despite some conservation of the internal gene arrangement cbb gene clusters of the various organisms differ in size and operon organization. The cbb operons of facultative autotrophs are more strictly regulated than those of obligate autotrophs. The major control is exerted by the cbbR gene, which codes for a transcriptional activator of the LysR family. This gene is typically located immediately upstream of and in divergent orientation to the regulated cbb operon, forming a control region for both transcriptional units. Recent studies suggest that additional protein factors are involved in the regulation. Although the metabolic signal(s) received by the regulatory components of the operons is (are) still unknown, the redox state of the cell is believed to play a key role. It is proposed that the control of the cbb operon expression is integrated into a regulatory network.

The two highly homologous cbb operons ofAlcaligenes eutrophus H16 that are located on the chromosome and on megaplasmid pHG1 contain genes encoding several enzymes of the Calvin carbon reduction cycle. Sequence analysis of a region from the promoter-distal part revealed two open reading frames, designated cbbT and cbbZ, at equivalent positions within the operons. Comparisons with known sequences suggested cbbT to encode transketolase (TK; EC 2.2.1.1) as an additional enzyme of the cycle. No significant overall sequence similarities were observed for cbbZ. Although both regions exhibited very high nucleotide identities, 93% (cbbZ) and 96% (cbbT), only the chromosomally encoded genes were heterologously expressed to high levels in Escherichia coli. The molecular masses of the observed gene products, CbbT (74 kDa) and CbbZ (24 kDa), correlated well with the values calculated on the basis of the sequence information. TK activities were strongly elevated in E. coli clones expressing cbbT, confirming the identity of the gene. Strains of E. coli harboring the chromosomal cbbZ gene showed high levels of activity of 2-phosphoglycolate phosphatase (PGP; EC 3.1.3.18), a key enzyme of glycolate metabolism in autotrophic organisms that is not present in wild-type E. coli. Derepression of the cbb operons during autotrophic growth resulted in considerably increased levels of TK activity and the appearance of PGP activity in A. eutrophus, although the pHG1-encoded cbbZ gene was apparently not expressed. To our knowledge, this study represents the first cloning and sequencing of a PGP gene from any organism.on July 5, 2020 by guest