Gene Expression System in Green Sulfur Bacteria by Conjugative Plasmid Transfer

Abstract

Gene transfer and expression systems in green sulfur bacteria were established by bacterial conjugation with Escherichia coli. Conjugative plasmid transfer from E. coli S17-1 to a thermophilic green sulfur bacterium, Chlorobaculum tepidum (formerly Chlorobium tepidum) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in C. tepidum cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, C. limnaeum (formerly Chlorobium phaeobacteroides) RK-j-1, was also achieved with pDSK5192. The expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the pscAB gene cluster on the C. tepidum genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆cycA and ∆soxB mutants were completely rescued after introduction of pDSK5191-cycA and -soxB that were designed to express their complementary genes. On the other hand, pDSK5191-6xhis-pscAB, which incorporated the gene cluster of 6xhis-pscA and pscB, produced approximately four times more of the photosynthetic reaction center complex with His-tagged PscA as compared with that expressed in the genome by the conventional natural transformation method. This expression system, based on conjugative plasmid, would be applicable to general molecular biological studies of green sulfur bacteria.

Mutation-induced perturbation of the special pair P840 in the homodimeric reaction center in green sulfur bacteria

“…To introduce H-bond interactions with P840 in the homodimeric GbRC, PscA-Leu688 and PscA-Val689 were substituted with cysteine residues by using the genetic tools that we have recently developed in C. tepidum 16 30 . These mutations, L688C and V689C, respectively, especially the former, were designed to mimic the interaction of PshA-Cys601 with P800 in HbRC.…”

“…We consider that a combination of two different factors would have induced these extremely low yields. First, the expression level of the 6xhis-psc A gene on the plasmid would be much higher (about 4- to 5-fold) than that of the strep- pscA gene on a chromosome, simply reflecting their copy numbers of about 9–12 for plasmid and 2–4 for chromosome, respectively, in C. tepidum 30 . Therefore, the probability of the dimerization between the His-tagged and Strep-tagged PscA polypeptides must be low.…”

N-methyl-bacillithiol, a new metabolite discovered in theChlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed

“…The Chlorobiaceae have contributed to our understanding of CO 2 fixation via the reductive TCA cycle (4) and light-harvesting mechanisms through studies of the chlorosome (5, 6), the light harvesting antenna in this group. Chlorobaculum tepidum (formerly Chlorobium tepidum ) is a model system for the Chlorobi because of its rapid growth rate (7), complete genome sequence (8), and genetic system (9–12).…”

Chlorobaculum tepidum Modulates Amino Acid Composition in Response to Energy Availability, as Revealed by a Systematic Exploration of the Energy Landscape of Phototrophic Sulfur Oxidation

“…Unlike the more metabolically versatile species of the family Chromatiaceae, which are capable of dark, aerobic chemotrophic growth in addition to phototrophic sulfur oxidation (16), all of the characterized members of the family Chlorobiaceae are obligate anaerobic photolithoautotrophs, although some Chlorobiaceae assimilate simple organic carbon compounds (acetate, pyruvate) (17). Chlorobaculum tepidum produces and degrades insoluble extracellular S 0 as an obligate intermediate of sulfide oxidation, grows rapidly (15,18), is genetically tractable (19)(20)(21)(22)(23), and has a sequenced genome (24), making it an ideal platform for studying S 0 metabolism by systems-based methods. Systems-based methods require reproducible growth under controlled conditions and robust methods of biomass quantitation, both of which are challenging for C. tepidum.…”