| Summary: | Two mutant strains of Rhodobacter capsulatus which lacked the heavy (H)
subunit of the reaction center (RC) were constructed. One strain, DW5, contains
a translationally in-frame deletion of the puhA gene, which encodes the RC H
subunit. The other strain, DW1, is a puhA polar mutation strain containing the
same deletion of the puhA gene coupled with the insertion of a
streptomydn/spectinomycin resistance omega (Ω) cartridge. Both strains were
unable to grow under photosynthetic conditions (PS ). Absorption spectroscopy
and SDS-PAGE analysis showed that these two strains have a reduction in the
amount of the light-harvesting I (LHI) complex. In DW5, both photosynthetic
growth and LHI complex level were restored by complementation in trans with
plasmid pPUHA, which contains the puhA gene. However, when pPUHA was
introduced into DW1, photosynthetic growth was only partially restored, and
the LHI complex level was not restored to the wild type phenotype. When DW1
was doubly complemented with pPUHA and pORF214/162b (which contains
orf214 and orfl62b, two open reading frames [orfs] downstream of puhA),
photosynthetic growth was restored to the wild type level. However, LHI
complex level was not restored in this doubly complemented strain. In a third
mutant strain, DW2, orf214 was directly mutated with an insertion of the Ω.
cartridge. DW2 also showed a PS" phenotype and a reduction in LHI complex
level. Interestingly, SDS-PAGE analysis showed that the RC H subunit was
missing in the intracytoplasmic membrane of DW2. Photosynthetic growth, but
not LHI complex level, was restored when DW2 was complemented with
pORFF214/162b. The roles of the RC H subunit and the gene products of the orfs
downstream of puhA in photosynthesis are discussed.
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