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Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

ABSTRACT: A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J. Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis. Similar diel patterns of carbon fixation and rbcL gene expression were observed in three of four time series, with maxima for photosynthetic capacity (P(max)) and rbcL mRNA occurring between 10 a.m. and 1 p.m. The lowest P(max) and rbcL levels were detected between 6 p.m. and 10:30 p.m. A significant correlation was found between P(max) and form ID rbcL mRNA (R(2) = 0.56) and forms IA and IB (R(2) = 0.41 and 0.47, respectively). The correlation between the abundance of "diatom" rbcL and P(max) mRNA was modest (R(2) = 0.49; n = 12) but improved dramatically (R(2) = 0.97; n = 10) upon removal of two outliers which represented afternoon samples with high P(max) but lower mRNA levels. Clone libraries from reverse transcription-PCR-amplified rbcL mRNA indicated the presence of several chromophytic algae (diatoms, prymnesiophytes, and chrysophytes) and some eukaryotic green flagellates. Analogous results were obtained from amplified small rRNA sequences and secondary pigment analysis. These results suggest that diatoms were a major contributor to carbon fixation at LEO 15 at the time of sampling and that photosynthetic carbon fixation was partially controlled by transcriptional regulation of the RubisCO gene.

SUBMITTER: Corredor JE

PROVIDER: S-EPMC520902 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

Corredor Jorge E JE Wawrik Boris B Paul John H JH Tran Hiep H Kerkhof Lee L López José M JM Dieppa Angel A Cárdenas Oswaldo O

Applied and environmental microbiology 20040901 9

A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J. Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis ...[more]

PMID: 15345433

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Project description:Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.

Project description:The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO(2) fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg(2+) for catalysis and, like RuBisCO, was stimulated by CO(2). Four residues that are essential for the enolization reaction of RuBisCO, Lys(175), Lys(201), Asp(203), and Glu(204), were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys(123), the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K(i) value of 103 mum. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.

Project description:The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Dataset Information

Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

Publications

Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

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