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Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina.

Chen GE, Hitchcock A, Jackson PJ, Chaudhuri RR, Dickman MJ, Hunter CN, Canniffe DP - J. Bacteriol. (2016)

Bottom Line: Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB.Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophyll d Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained both bciA and bciB.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Construction of Synechocystis strains designed to express putative A. marina 8VR-encoding genes. (A) Isolation of fully segregated Synechocystis ΔbciB strains containing genes from A. marina, confirmed by colony PCR amplifying the psbAII locus. (B) Expression of recombinant proteins was confirmed by resolving membrane fractions from the described strains by SDS-PAGE, transferring to a membrane, and probing with anti-BciB and anti-His6 antibodies.
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Figure 2: Construction of Synechocystis strains designed to express putative A. marina 8VR-encoding genes. (A) Isolation of fully segregated Synechocystis ΔbciB strains containing genes from A. marina, confirmed by colony PCR amplifying the psbAII locus. (B) Expression of recombinant proteins was confirmed by resolving membrane fractions from the described strains by SDS-PAGE, transferring to a membrane, and probing with anti-BciB and anti-His6 antibodies.

Mentions: ORFs encoding proteins with high sequence similarity to both BciA and BciB found in the genome sequence of A. marina (26) are annotated as genes encoding a transcriptional regulator (nmrA) and a subunit of a NiFe hydrogenase, responsible for coenzyme F420 reduction in archaeal methanogenesis (frhB), respectively. In order to test the possible 8VR activities of the encoded proteins, nmrA and frhB were expressed in a ΔbciB mutant of Synechocystis (11) that is unable to synthesize 8E-Chl a and, as a consequence, is unable to grow under high-light conditions (13, 14). The A. marina genes were integrated into the genome of Synechocystis ΔbciB in place of the psbAII gene (one of three ORFs encoding the D1 protein of photosystem II; deletion of a single copy of the gene does not affect photosynthetic capability [37]) (Fig. 2A). To determine whether the recombinant proteins were produced in Synechocystis, samples from cultures of wild-type (WT), ΔbciB, ΔbciB::nmrA(Am) and ΔbciB::frhB(Am) strains, grown under moderate light intensity, were disrupted by bead beating, and the soluble and membrane fractions were separated by centrifugation. The membrane fractions were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, which was probed with an antibody raised against Synechocystis BciB and, in the absence of an antibody raised against BciA, a commercial anti-His6 antibody (Bethyl Laboratories, Inc.) (Fig. 2B). The blot indicates that the recombinant proteins are present, confirming the effective expression of the A. marina genes when under the control of the psbAII promoter.


Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina.

Chen GE, Hitchcock A, Jackson PJ, Chaudhuri RR, Dickman MJ, Hunter CN, Canniffe DP - J. Bacteriol. (2016)

Construction of Synechocystis strains designed to express putative A. marina 8VR-encoding genes. (A) Isolation of fully segregated Synechocystis ΔbciB strains containing genes from A. marina, confirmed by colony PCR amplifying the psbAII locus. (B) Expression of recombinant proteins was confirmed by resolving membrane fractions from the described strains by SDS-PAGE, transferring to a membrane, and probing with anti-BciB and anti-His6 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836224&req=5

Figure 2: Construction of Synechocystis strains designed to express putative A. marina 8VR-encoding genes. (A) Isolation of fully segregated Synechocystis ΔbciB strains containing genes from A. marina, confirmed by colony PCR amplifying the psbAII locus. (B) Expression of recombinant proteins was confirmed by resolving membrane fractions from the described strains by SDS-PAGE, transferring to a membrane, and probing with anti-BciB and anti-His6 antibodies.
Mentions: ORFs encoding proteins with high sequence similarity to both BciA and BciB found in the genome sequence of A. marina (26) are annotated as genes encoding a transcriptional regulator (nmrA) and a subunit of a NiFe hydrogenase, responsible for coenzyme F420 reduction in archaeal methanogenesis (frhB), respectively. In order to test the possible 8VR activities of the encoded proteins, nmrA and frhB were expressed in a ΔbciB mutant of Synechocystis (11) that is unable to synthesize 8E-Chl a and, as a consequence, is unable to grow under high-light conditions (13, 14). The A. marina genes were integrated into the genome of Synechocystis ΔbciB in place of the psbAII gene (one of three ORFs encoding the D1 protein of photosystem II; deletion of a single copy of the gene does not affect photosynthetic capability [37]) (Fig. 2A). To determine whether the recombinant proteins were produced in Synechocystis, samples from cultures of wild-type (WT), ΔbciB, ΔbciB::nmrA(Am) and ΔbciB::frhB(Am) strains, grown under moderate light intensity, were disrupted by bead beating, and the soluble and membrane fractions were separated by centrifugation. The membrane fractions were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, which was probed with an antibody raised against Synechocystis BciB and, in the absence of an antibody raised against BciA, a commercial anti-His6 antibody (Bethyl Laboratories, Inc.) (Fig. 2B). The blot indicates that the recombinant proteins are present, confirming the effective expression of the A. marina genes when under the control of the psbAII promoter.

Bottom Line: Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB.Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophyll d Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained both bciA and bciB.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.

No MeSH data available.


Related in: MedlinePlus