A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress

Abstract

A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum. A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp. thiosulfatophilum strain Tassajara. Analysis of these amino acid sequences indicated that they lacked several conserved RubisCO active site residues. The Chlorobium RubisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis and Archaeoglobus fulgidus, which also lack some typical RubisCO active site residues. When the C. tepidum gene encoding the RubisCO-like protein was disrupted, the resulting mutant strain displayed a pleiotropic phenotype with defects in photopigment content, photoautotrophic growth and carbon fixation rates, and sulfur metabolism. Most important, the mutant strain showed substantially enhanced accumulation of two oxidative stress proteins. These results indicated that the C. tepidum RubisCO-like protein might be involved in oxidative stress responses and/or sulfur metabolism. This protein might be an evolutional link to bona fide RubisCO and could serve as an important tool to analyze how the RubisCO active site developed.

Figure 1

Phylogenetic tree of RubisCO (Forms I-III) and RubisCO-like amino acid sequences (Form IV) from various organisms. Multiple sequence alignments and trees were produced with clustal x and visualized with tree view 1.4. Bootstrap values indicated at the nodes are the percentage of times that particular node appeared in 1,000 trials.

Figure 2

Partial amino acid sequence alignment of form I RubisCO (Synechococcus sp. strain PCC 6301; I-Syn 6301), form II RubisCO (Rhodospirillum rubrum; II-Rr), form III RubisCO (Methanococcus jannaschii; III-Mj) and form IV RLPs from Archaeoglobus fulgidus (IV-Af-RbcL-1), Bacillus subtilis (IV-Bs-YkrW), C. tepidum (IV-Ct-RLP) and C. limicola (IV-Cl-RLP). Active site residues are highlighted in black. Residues deviating from the active site consensus are highlighted in gray. C, residues involved in catalysis; R, residues involved in RuBP binding. The alignment is numbered according to the Synechococcus sp. strain PCC 6301 sequence.

Figure 3

Physical map of the C. tepidum genome surrounding the gene encoding the RLP and strategy for mutant construction. The closest homolog for each ORF was identified by blastp searches of the nonredundant protein database at the National Center for Biotechnology Information. Percent identity and similarity are at the amino acid level. The positions of the Ω cassette insertions are designated by the name of the resulting mutant strain.

Figure 4

Elemental sulfur levels in early stationary phase (≈220 μg protein ml⁻¹) autotrophic cultures of WT2321 and strain Ω∷ RLP. Media additions: Cys, 0.5 mM cysteine; Ser, 0.5 mM serine.

Figure 5

SDS/PAGE analysis of 20 μg of cell extract protein from WT2321 (WT) or the Ω∷ RLP mutant strain (Ω∷ RLP). CO₂, photoautotrophic growth; Cys, photoautotrophic + 0.5 mM cysteine; Ser, photoautotrophic + 0.5 mM serine. Each lane contains cell extract from independent cultures harvested after 16 h of growth.

Figure 6

Superoxide dismutase activity in crude extracts of WT2321 (wild type), strain Ω∷ RLP (RLP) and strain Ω∷SDR (SDR). Data represent the mean ± standard deviation of three independent cultures for each strain. One unit of specific activity corresponds to the amount of protein required to inhibit 50% of the rate of cytochrome c reduction by xanthine oxidase in an extract free standard assay (26).