Complementation analysis and regulation of CO2 fixation gene expression in a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum

Abstract

A ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain ofRhodospirillum rubrum that was incapable of photolithoautotrophic growth was constructed. Photoheterotrophic growth, however, was possible for the R. rubrum RubisCO deletion strain when oxidized carbon compounds such as malate were supplied. The R. rubrum RubisCO-deficient strain was not complemented to photolithoautotrophic growth by various R. rubrum DNA fragments that contain the gene encoding RubisCO, cbbM. When the R. rubrum cbbM deletion strain harbored plasmids containing R. rubrum DNA inserts with at least 2.0 kb preceding the translational start site of the cbbM gene, RubisCO activity and RubisCO antigen were detected. Lack of RubisCO expression was therefore not the cause for the failure to complement the cbbM mutant strain. Interestingly, DNA fragments encoding either of two complete Calvin-Benson-Bassham C02 fixation (cbb) gene operons from Rhodobacter sphaeroides were able to complement the R. rubrum RubisCO deletion strain to photolithoautotrophic growth. The same R. rubrum DNA fragments that failed to complement the R. rubrum cbbM deletion strain successfully complemented the RubisCO deletion strain of R. sphaeroides, pointing to distinct differences in the regulation of metabolism and the genetics of photolithoautotrophic growth in these two organisms. A number of cbb genes were identified by nucleotide sequence analysis of the region upstream of cbbM. Included among these was an open reading frame encoding a cbbR gene showing a high degree of sequence similarity to known lysR-type CO2 fixation transcriptional activator genes. The placement and orientation of the cbbR transcriptional regulator gene in R. rubrum are unique.

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways

“…Strain I19A (⌬cbbM) is a derivative of the WT strain in which the Rubisco gene (cbbM) is disrupted by a kanamycin marker (17). Strain IR (⌬cbbM/⌬rlpA) is a derivative of I19A in which the RLP gene (rlpA) is disrupted by a gentamycin marker (14).…”

“…Bacterial Growth and Complementation Studies-Bacterial conjugation for complementation studies was performed by biparental mating as described previously (14,17), using Escherichia coli strain SM-10 as the donor strain. All plasmids employed contained a tetracycline resistance marker that was used for counter-selection of transconjugants.…”

“…2, A and B) (14). However, deletion of the form II Rubisco (cbbM) gene (strains I19A (⌬cbbM) (17) and IR (⌬cbbM/⌬rlpA) (14)) resulted in the inability of the organism to allow significant photoheterotrophic growth on MTA as sole sulfur source, with or without the presence of a functional RLP (Fig. 2, B and D) (14).…”

“…2C). Rubisco knock-out strains of R. rubrum are incapable of photoautotrophic growth (17,28) and grow poorly under photoheterotrophic conditions using substrates such as malate as electron donor and carbon source and sulfate as the sulfur source (Fig. 2, Table 1).…”

The role of two CbbRs in the transcriptional regulation of three ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Hydrogenovibrio marinus strain MH-110

“…CbbR, one of the LysR-type transcriptional regulators (LTTRs), has been found in many photo-and chemoautotrophic bacteria, and has been shown to be required to activate expression of RubisCO (Windhövel & Bowien, 1991;Falcone & Tabita, 1993;Gibson & Tabita, 1993;van den Bergh et al, 1993;Paoli et al, 1998;Dubbs et al, 2000Dubbs et al, , 2004Vichivanives et al, 2000). Although the cbbR genes are located upstream of genes encoding RubisCO in a divergent orientation in most bacteria, the number of cbbR genes is not equivalent to that of genes for RubisCO (Windhövel & Bowien, 1991;Gibson & Tabita, 1993;Meijer et al, 1996;Kusian & Bowien, 1997;Smith & Tabita, 2002;Dubbs & Tabita, 2003).…”