Cloning and expression of the ?-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Abstract

Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides, respectively. However, only the previously reported form I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A SalI fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5 alpha. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus. Other Calvin cycle genes were not clustered with either the form I or form II genes.

Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats

“…This has been shown previously for other SOB species, such as Acidithiobacillus ferrooxidans, Thiobacillus sp. (English et al, 1992), Halothiobacillus neapolitanus (Baker et al, 1998), Thiomonas intermedia (Stoner & Shively, 1993) and Thiomicrospira sp. (Tourova et al, 2006).…”

Occurrence, phylogeny and evolution of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in obligately chemolithoautotrophic sulfur-oxidizing bacteria of the genera Thiomicrospira and Thioalkalimicrobium

“…Cupriavidus necator H16 (formerly Alcaligenes eutrophus H16) possesses two sets of almost identical genes which encode the red-like form I enzyme (Kusian et al, 1995), Allochromatium vinosum (formerly Chromatium vinosum) has two sets of divergent genes which encode the green-like form I enzyme (Viale et al, 1989) and Rhodobacter azotoformans has two sets of genes which encode both green-and red-like form I enzymes (Uchino & Yokota, 2003). Furthermore, Halothiobacillus neapolitanus (formerly Thiobacillus neapolitanus), Thiomonas intermedia (formerly Thiobacillus intermedius), Thiobacillus denitrificans, Rhodobacter sphaeroides and Rhodobacter capsulatus have genes for both form I and form II enzymes (Gibson & Tabita, 1977a, b;Shively et al, 1986;English et al, 1992;Stoner & Shively, 1993;Paoli et al, 1995). Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) and Hydrogenovibrio marinus have three different sets of RubisCO genes, two encoding the green-like form I enzyme, whereas the third one encodes a form II enzyme (Kusano et al, 1991;Heinhorst et al, 2002;Yaguchi et al, 1994;Nishihara et al, 1998).…”

Organization of Carboxysome Genes in the Thiobacilli

“…intermedia K12 Sau3A-lambda library with the cbbL gene from A. nidulans resulted in the isolation of three positive clones designated Ti102, Ti104, and Ti108 [27,28]. Probing these clones with the csoS1 gene of H. neapolitanus demonstrated that Ti102 and Ti108, but not Ti104, contained at least parts of both cbbL and csoS1 genes (unpublished).…”