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Gene expression system in green sulfur bacteria by conjugative plasmid transfer.

Azai C, Harada J, Hirozo OO - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.

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.

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Absorption spectra of BChl a-associated proteins from various C. tepidum strains recovered by Ni2+-affinity purification.Absorption spectra (traces 1-6) of BChl a-associated proteins, which were obtained from various C. tepidum strains with Ni2+-affinity chromatography purification procedure, were measured (see Materials and Methods). Each spectrum was normalized so that recovery rates per wet cell weights can be compared. In the inset, spectra of the eluates from the wild-type WT2321 strain (trace 1) and the 6HA-∆recA strain with pDSK5191-6xhis-pscAB (trace 5) were normalized at absorption peaks around 810 nm. The absorption band around 810 nm is contributed by antenna BChls a associated with FMO proteins and the RC complex as well, while the absorption shoulder around 840 nm is specific to a special pair of BChls a, P840, within the RC complex [3,13].
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pone-0082345-g004: Absorption spectra of BChl a-associated proteins from various C. tepidum strains recovered by Ni2+-affinity purification.Absorption spectra (traces 1-6) of BChl a-associated proteins, which were obtained from various C. tepidum strains with Ni2+-affinity chromatography purification procedure, were measured (see Materials and Methods). Each spectrum was normalized so that recovery rates per wet cell weights can be compared. In the inset, spectra of the eluates from the wild-type WT2321 strain (trace 1) and the 6HA-∆recA strain with pDSK5191-6xhis-pscAB (trace 5) were normalized at absorption peaks around 810 nm. The absorption band around 810 nm is contributed by antenna BChls a associated with FMO proteins and the RC complex as well, while the absorption shoulder around 840 nm is specific to a special pair of BChls a, P840, within the RC complex [3,13].

Mentions: The expression of His-tagged PscA was confirmed by Ni2+-affinity purification of the His-tagged RC complex (Figure 4). The eluates obtained from the wild-type and ∆recA mutant strains were pale green and showed their Qy-peak wavelengths at 809 nm (Figure 4, traces 1 and 2). As these spectra did not have any absorption shoulder around 840 nm (Figure 4, inset), which serves as a criterion for the presence of the RC complex [3,13], their eluates would contain FMO proteins adsorbed nonspecifically to the Ni2+ resin. On the other hand, the specific adsorption of the His-tagged RC complex was observed in the case of the C. tepidum 6HA strain (Figure 4, trace 3), whose authentic pscA gene on the chromosome was replaced with the 6xhis-pscA gene (see Materials and Methods). The same result was obtained in the case of the 6HA-∆recA strain (Figure 4, trace 4), which was a recA-deletion mutant derived from the 6HA strain. When introducing plasmid pDSK5191-6xhis-pscAB into the ∆recA mutant (Figure 4, trace 6), the specific adsorption of the His-tagged RC complex was also observed, and in significantly larger amounts than that of the 6HA and 6HA-∆recA strains. Since the eluate from the ∆recA mutant did not contain any His-tagged RC complex, this result clearly indicates that the His-tagged PscA polypeptide was stably expressed from plasmid pDSK5191-6xhis-pscAB in the ∆recA mutant cells. Moreover, much larger amounts of the His-tagged RC complex were obtained by introducing plasmid pDSK5191-6xhis-pscAB into the 6HA-∆recA strain (Figure 4, trace 5). There was still a significant increase in the amount of the His-tagged RC complexes recovered from the 6HA-∆recA strain with plasmid pDSK5191-6xhis-pscAB as compared to the strain from the ∆recA mutant with the same plasmid, indicating that the 6xhis-pscA gene on the chromosome never suppressed the expression of its counterpart on the plasmid.


Gene expression system in green sulfur bacteria by conjugative plasmid transfer.

Azai C, Harada J, Hirozo OO - PLoS ONE (2013)

Absorption spectra of BChl a-associated proteins from various C. tepidum strains recovered by Ni2+-affinity purification.Absorption spectra (traces 1-6) of BChl a-associated proteins, which were obtained from various C. tepidum strains with Ni2+-affinity chromatography purification procedure, were measured (see Materials and Methods). Each spectrum was normalized so that recovery rates per wet cell weights can be compared. In the inset, spectra of the eluates from the wild-type WT2321 strain (trace 1) and the 6HA-∆recA strain with pDSK5191-6xhis-pscAB (trace 5) were normalized at absorption peaks around 810 nm. The absorption band around 810 nm is contributed by antenna BChls a associated with FMO proteins and the RC complex as well, while the absorption shoulder around 840 nm is specific to a special pair of BChls a, P840, within the RC complex [3,13].
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842273&req=5

pone-0082345-g004: Absorption spectra of BChl a-associated proteins from various C. tepidum strains recovered by Ni2+-affinity purification.Absorption spectra (traces 1-6) of BChl a-associated proteins, which were obtained from various C. tepidum strains with Ni2+-affinity chromatography purification procedure, were measured (see Materials and Methods). Each spectrum was normalized so that recovery rates per wet cell weights can be compared. In the inset, spectra of the eluates from the wild-type WT2321 strain (trace 1) and the 6HA-∆recA strain with pDSK5191-6xhis-pscAB (trace 5) were normalized at absorption peaks around 810 nm. The absorption band around 810 nm is contributed by antenna BChls a associated with FMO proteins and the RC complex as well, while the absorption shoulder around 840 nm is specific to a special pair of BChls a, P840, within the RC complex [3,13].
Mentions: The expression of His-tagged PscA was confirmed by Ni2+-affinity purification of the His-tagged RC complex (Figure 4). The eluates obtained from the wild-type and ∆recA mutant strains were pale green and showed their Qy-peak wavelengths at 809 nm (Figure 4, traces 1 and 2). As these spectra did not have any absorption shoulder around 840 nm (Figure 4, inset), which serves as a criterion for the presence of the RC complex [3,13], their eluates would contain FMO proteins adsorbed nonspecifically to the Ni2+ resin. On the other hand, the specific adsorption of the His-tagged RC complex was observed in the case of the C. tepidum 6HA strain (Figure 4, trace 3), whose authentic pscA gene on the chromosome was replaced with the 6xhis-pscA gene (see Materials and Methods). The same result was obtained in the case of the 6HA-∆recA strain (Figure 4, trace 4), which was a recA-deletion mutant derived from the 6HA strain. When introducing plasmid pDSK5191-6xhis-pscAB into the ∆recA mutant (Figure 4, trace 6), the specific adsorption of the His-tagged RC complex was also observed, and in significantly larger amounts than that of the 6HA and 6HA-∆recA strains. Since the eluate from the ∆recA mutant did not contain any His-tagged RC complex, this result clearly indicates that the His-tagged PscA polypeptide was stably expressed from plasmid pDSK5191-6xhis-pscAB in the ∆recA mutant cells. Moreover, much larger amounts of the His-tagged RC complex were obtained by introducing plasmid pDSK5191-6xhis-pscAB into the 6HA-∆recA strain (Figure 4, trace 5). There was still a significant increase in the amount of the His-tagged RC complexes recovered from the 6HA-∆recA strain with plasmid pDSK5191-6xhis-pscAB as compared to the strain from the ∆recA mutant with the same plasmid, indicating that the 6xhis-pscA gene on the chromosome never suppressed the expression of its counterpart on the plasmid.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.

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.

Show MeSH
Related in: MedlinePlus