• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Apr 1996; 178(8): 2334–2342.
PMCID: PMC177942

Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes.

Abstract

Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

Full Text

The Full Text of this article is available as a PDF (379K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams CW, Forrest ME, Cohen SN, Beatty JT. Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon. J Bacteriol. 1989 Jan;171(1):473–482. [PMC free article] [PubMed]
  • Allen JP, Feher G, Yeates TO, Komiya H, Rees DC. Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6162–6166. [PMC free article] [PubMed]
  • Allen JP, Feher G, Yeates TO, Komiya H, Rees DC. Structure of the reaction center from Rhodobacter sphaeroides R-26: protein-cofactor (quinones and Fe2+) interactions. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8487–8491. [PMC free article] [PubMed]
  • Bauer CE, Buggy JJ, Yang ZM, Marrs BL. The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts. Mol Gen Genet. 1991 Sep;228(3):433–444. [PubMed]
  • Bérard J, Gingras G. The puh structural gene coding for the H subunit of the Rhodospirillum rubrum photoreaction center. Biochem Cell Biol. 1991 Feb-Mar;69(2-3):122–131. [PubMed]
  • Chang CH, el-Kabbani O, Tiede D, Norris J, Schiffer M. Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides. Biochemistry. 1991 Jun 4;30(22):5352–5360. [PubMed]
  • Chory J, Donohue TJ, Varga AR, Staehelin LA, Kaplan S. Induction of the photosynthetic membranes of Rhodopseudomonas sphaeroides: biochemical and morphological studies. J Bacteriol. 1984 Aug;159(2):540–554. [PMC free article] [PubMed]
  • Davison J, Heusterspreute M, Chevalier N, Ha-Thi V, Brunel F. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene. 1987;51(2-3):275–280. [PubMed]
  • Deisenhofer J, Michel H. Structures of bacterial photosynthetic reaction centers. Annu Rev Cell Biol. 1991;7:1–23. [PubMed]
  • Dierstein R. Synthesis of pigment-binding protein in toluene-treated Rhodopseudomonas capsulata and in cell-free systems. Eur J Biochem. 1984 Feb 1;138(3):509–518. [PubMed]
  • Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang XW, Finlay DR, Guiney D, Helinski DR. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. [PubMed]
  • Ermler U, Fritzsch G, Buchanan SK, Michel H. Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution: cofactors and protein-cofactor interactions. Structure. 1994 Oct 15;2(10):925–936. [PubMed]
  • Farchaus JW, Barz WP, Grünberg H, Oesterhelt D. Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides. EMBO J. 1992 Aug;11(8):2779–2788. [PMC free article] [PubMed]
  • Farchaus JW, Gruenberg H, Oesterhelt D. Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype. J Bacteriol. 1990 Feb;172(2):977–985. [PMC free article] [PubMed]
  • Johnson JA, Wong WK, Beatty JT. Expression of cellulase genes in Rhodobacter capsulatus by use of plasmid expression vectors. J Bacteriol. 1986 Aug;167(2):604–610. [PMC free article] [PubMed]
  • Karrasch S, Bullough PA, Ghosh R. The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J. 1995 Feb 15;14(4):631–638. [PMC free article] [PubMed]
  • Kiley PJ, Kaplan S. Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol Rev. 1988 Mar;52(1):50–69. [PMC free article] [PubMed]
  • Klug G, Cohen SN. Pleiotropic effects of localized Rhodobacter capsulatus puf operon deletions on production of light-absorbing pigment-protein complexes. J Bacteriol. 1988 Dec;170(12):5814–5821. [PMC free article] [PubMed]
  • LeBlanc HN, Beatty JT. Rhodobacter capsulatus puc operon: promoter location, transcript sizes and effects of deletions on photosynthetic growth. J Gen Microbiol. 1993 Jan;139(1):101–109. [PubMed]
  • Lilburn TG, Haith CE, Prince RC, Beatty JT. Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus. Biochim Biophys Acta. 1992 May 20;1100(2):160–170. [PubMed]
  • Lilburn TG, Prince RC, Beatty JT. Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype. J Bacteriol. 1995 Aug;177(16):4593–4600. [PMC free article] [PubMed]
  • Nieth KF, Drews G, Feick R. Photochemical reaction centers from Rhodopseudomonas capsulata. Arch Microbiol. 1975 Sep 30;105(1):43–45. [PubMed]
  • Peterson GL. Determination of total protein. Methods Enzymol. 1983;91:95–119. [PubMed]
  • Pollock D, Bauer CE, Scolnik PA. Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter. Gene. 1988 May 30;65(2):269–275. [PubMed]
  • Prentki P, Krisch HM. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. [PubMed]
  • Prince RC, Dutton PL, Clayton BJ, Clayton RK. EPR properties of the reaction center of Rhodopseudomas gelatinosa in situ and in a detergent-solubilized form. Biochim Biophys Acta. 1978 May 10;502(2):354–358. [PubMed]
  • Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. [PubMed]
  • Scolnik PA, Zannoni D, Marrs BL. Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata. Biochim Biophys Acta. 1980 Dec 3;593(2):230–240. [PubMed]
  • Snozzi M, Bachofen R. Characterisation of reaction centers and their phospholipids from Rhodospirillum rubrum. Biochim Biophys Acta. 1979 May 9;546(2):236–247. [PubMed]
  • Sockett RE, Donohue TJ, Varga AR, Kaplan S. Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences. J Bacteriol. 1989 Jan;171(1):436–446. [PMC free article] [PubMed]
  • Solioz M, Marrs B. The gene transfer agent of Rhodopseudomonas capsulata. Purification and characterization of its nucleic acid. Arch Biochem Biophys. 1977 May;181(1):300–307. [PubMed]
  • Taylor DP, Cohen SN, Clark WG, Marrs BL. Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique. J Bacteriol. 1983 May;154(2):580–590. [PMC free article] [PubMed]
  • Varga AR, Kaplan S. Synthesis and stability of reaction center polypeptides and implications for reaction center assembly in Rhodobacter sphaeroides. J Biol Chem. 1993 Sep 15;268(26):19842–19850. [PubMed]
  • Weaver PF, Wall JD, Gest H. Characterization of Rhodopseudomonas capsulata. Arch Microbiol. 1975 Nov 7;105(3):207–216. [PubMed]
  • Williams JC, Steiner LA, Feher G. Primary structure of the reaction center from Rhodopseudomonas sphaeroides. Proteins. 1986 Dec;1(4):312–325. [PubMed]
  • Yen HC, Hu NT, Marrs BL. Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata. J Mol Biol. 1979 Jun 25;131(2):157–168. [PubMed]
  • Youvan DC, Hearst JE, Marrs BL. Isolation and characterization of enhanced fluorescence mutants of Rhodopseudomonas capsulata. J Bacteriol. 1983 May;154(2):748–755. [PMC free article] [PubMed]
  • Zilsel J, Lilburn TG, Beatty JT. Formation of functional inter-species hybrid photosynthetic complexes in Rhodobacter capsulatus. FEBS Lett. 1989 Aug 14;253(1-2):247–252. [PubMed]
  • Zsebo KM, Hearst JE. Genetic-physical mapping of a photosynthetic gene cluster from R. capsulata. Cell. 1984 Jul;37(3):937–947. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...