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Woronowicz K Olubanjo OB Sung HC Lamptey JL Niederman RA 《Photosynthesis research》2012,111(1-2):125-138
In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band. 相似文献
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Woronowicz K Olubanjo OB Sung HC Lamptey JL Niederman RA 《Photosynthesis research》2011,108(2-3):201-214
In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band. 相似文献
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Characterization and complementation of a mutant of Rhodobacter sphaeroides with a chromosomal deletion in the light-harvesting (LH2) genes 总被引:3,自引:0,他引:3
An LH2- strain of Rhodobacter sphaeroides, DBC1, has been constructed by deleting the puc operon, which encodes the LH2 alpha and beta polypeptides, from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1.25 kb Km(R) cassette derived from Tn903. Strain DBC1 lacked the LH2 complex, as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3.8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides. 相似文献
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Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. 总被引:20,自引:7,他引:13
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Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect. 相似文献
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Zhao Z Hu Z Nie X Cheng L Ding G Luo M Pan Y Liang Y Chen G 《Protein and peptide letters》2011,18(6):568-572
Heterologous protein expression levels can not be evaluated in real-time by experimental procedures commonly used for most expression systems during host cell culture. Rb. sphaeroides has provided an ideal system for studying both photosynthesis and membrane development and exhibited potential as a novel expression system. We constructed the puc1BA and puc2BA mutant strain Rb. sphaeroides CQU68 and used it as a novel expression system to heterologously express proteins fused to β-subunit of light-harvesting 2 complexes (LH2). The presence of LH2 with β-subunit fusion proteins was spectrally detected by the LH2 typical absorption at ~800 nm and ~850 nm, and the formation of these complexes were further confirmed by SDS-PAGE and Western blot analysis. The expression levels of heterologous protein measured by SDS-PAGE and Western blot turned out to be higher as the typical spectral peak heights increase. These findings suggested that the production of the heterologous protein could be rapidly detected through the LH2 absorption at ~800 nm and ~850 nm. Moreover, the typical absorption could be used as a monitor for rapid and real-time evaluation of heterologous protein expression levels. 相似文献
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C L Turnbough K H Kerr W R Funderburg J P Donahue F E Powell 《The Journal of biological chemistry》1987,262(21):10239-10245
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