Investigation of Rhodobacter capsulatus PufX interactions in the core complex of the photosynthetic apparatus

The photosynthetic apparatus of purple bacteria in the genus Rhodobacter includes a core complex consisting of the reaction centre (RC), light-harvesting complex 1 (LH1), and the PufX protein. PufX modulates LH1 structure and facilitates photosynthetic quinone/quinol exchange. We deleted RC/LH1 genes in pufX ⁺ and pufX ⁺⁺ (merodiploid) strains of Rhodobacter capsulatus, which reduced PufX levels regardless of pufX gene copy number and location. Photosynthetic growth of RC-only strains and independent assembly kinetics of the RC and LH1 were unaffected by pufX merodiploidy, but the absorption spectra of strains expressing the RC plus either LH1 α or β indicated that PufX may influence bacteriochlorophyll binding environments. Significant self-association of the PufX transmembrane segment was detected in a hybrid protein expression system, consistent with a role of PufX in core complex dimerization, as proposed for other Rhodobacter species. Our results indicate that in R. capsulatus PufX has the potential to be a central, homodimeric core complex component, and its cellular level is increased by interactions with the RC and LH1.     

Molecular phylogeny of theChironomus genus deduced from nucleotide sequences of two nuclear genes,ssp 160 and the globin 2b gene

Two genes were employed to study phylogenetic relatedness of theChironomus species: the protein-coding, salivary gland-specificssp160 gene, and the globin 2b (gb2b) gene. By using PCR, it was demonstrated that all the 38Chironomus species analyzed possess thegb2b gene, while only 13 have thessp160 gene. Partial nucleotide sequences of the genes of 22 species were determined. The data obtained were employed to construct phylogenetic trees which appeared to be topologically similar and revealed five groups of phylogenetically closely related species. Combining the data obtained in the studies of nuclear and mitochondrial genes, a molecular-data-based scenario could be suggested for theChironomus genus evolution.     

Immunocytochemical localization and characterization of mammalian prolactin- and somatotropin-like material in the pituitary of the Australian lungfish,Neoceratodus forsteri

The aim of the present study was to define at the light-microscopic level expression of prolactin and somatotropin material in the pituitary gland of the Australian lungfish, Neoceratodus forsteri, by use of polyclonal antibodies against ovine prolactin (oPRL) and bovine somatotropin (bSTH). Substances immunologically related to mammalian oPRL as well as bSTH were detected in two morphologically different cell types in the distal lobe, corresponding to the acidophilic cells. The specificity of the antibodies was initially confirmed in a porcine tissue control system. First, our absorption studies confirm that in Neoceratodus the anti-oPRL identifies part of an oPRL-like molecule different from bSTH. Secondly, the anti-bSTH identifies both part of a bSTH-like molecule proper to bovine and Neoceratodus STH, and part of a bSTH-like molecule having antigenic determinants in common with both bSTH and oPRL. This part of the oPRL is, however, not shared with the Neoceratodus PRL as revealed by the anti-oPRL. Altogether these observations support the concepts: (1) that mammalian PRL and STH, or part of those, were established early in evolution, and (2) that dipnoans as living sarcopterygians have an ancestor in common with the early amphibians. The exact nature and physiological functions of the substances detected remain to be defined.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Expression of antimicrobial peptide LH multimers in <Emphasis Type="Italic">Escherichia coli</Emphasis> C43(DE3)

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni²⁺-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC₅₀ of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper.     

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy

Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Q_y absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E _Δ ≈ 3.4 × 10⁵ [V/cm]. This change can explain the 3 nm wavelength shift of the Q_y absorption band in the reconstituted LH1 complex.     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda

Summary The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types are recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH (1–24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.     

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc ₁ complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc ₁ complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.     

Deletions of the YNZ22and Alu-VpA/MycL1Loci and Post-Surgery Prognosis in Human Colorectal Adenocarcinoma

Since deletions of the short arm of chromosome 17 are the most common genetic defects in human colorectal carcinoma (CC), we tested the YNZ22locus (D17S30, 17p13.3) for loss of heterozygosity (LH) in adenocarcinoma and in the normal colonic mucosa of 49 CC patients, and studied the association of LH with clinicomorphological features of the tumor. Allele frequency distribution of YNZ22did not differ for the patients and healthy people. LH in YNZ22in the tumor was found in 33% (13/39) of all informative cases, its frequency being thrice higher in men than in women (²= 5.21, p= 0.022). The defect was associated with moderate or poor histological differentiation (P ₂= 0.0055) and polyploidy >3n(P ₂= 0.0035) of tumor cells and with high incidence of post-surgery relapse or metastasis. Analysis of both YNZ22and Alu-VpA/MycL1(1p34.3) loci in the tumor allowed reliable relapse prognosis in 76% of the CC patients. The probability of post-surgery relapse or metastasis was estimated at no less than 67% for patients with LH in at least one of the two loci in the tumor, and at somewhat more than 20% for patients without LH.     

Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol

Peptide:N-glycanase (PNGase) releases N-glycans from glycoproteins/glycopeptides. Cytoplasmic PNGase is widely recognized as a component of machinery for ER-associated degradation (ERAD), i.e. proteasomal degradation of misfolded, newly synthesized (glyco)proteins that have been exported from the ER. The enzyme belongs to the “transglutaminase superfamily” that contains a putative catalytic triad of cysteine, histidine, and aspartic acid. The mammalian orthologues of PNGase contain the N-terminal PUB domain that serves as the protein–protein interaction domain. The C-terminus of PNGase was recently found to be a novel carbohydrate-binding domain. Taken together, these observations indicate that C-terminus of mammalian PNGase is important for recognition of the substrates while N-terminus of this enzyme is involved in assembly of a degradation complex.     

Photoperiodic activation of Fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail

Photoperiodic stimulation of quail (Coturnix coturnix japonica) resulted in the appearance of a nuclear fos-like protein within neurones of the basal tuberal hypothalamus. On transfer to long days the number of neurones containing this fos-like immunoreactivity increased from about 150 to 700, the neurones being scattered throughout the length of the tubero-infundibular complex. This activation had occurred by early in the second long day and was maintained for at least three long days. Over this period circulating levels of LH increased seven-fold, indicating that photoperiodic induction had taken place in the birds. A similar time-course of fos-like induction occurred in castrated quail exposed to a single long day and then returned to short days. Activation mirrored the long-term changes in LH secretion found in this paradigm and fos-like immunoreactivity showed the same carry-over characteristics of photoperiodic induction, being maximal two days after the quail had been exposed to the single long day (and were again on short days) and when LH secretion was at its maximum. Activation of fos-like immunoreactive cells did not take place when long-day quail were transferred to short photoperiods. The evidence supports the view that the neurones being activated are involved in a specific fashion in the avian photoperiodic response.     

The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: Comparative analyses of light-harvesting proteins and the cytochrome subunits associated with the reaction centers

The nucleotide sequence of the puf operon of the purple bacterium, Rhodospirillum molischianum, was determined. The operon includes genes coding for the and subunits of the light-harvesting 1 (LH1) complex and the L, M, and cytochrome subunits of the reaction center complex. As in other purple bacteria, the genes are arranged within the operon in this order. As in Rubrivivax gelatinosus, the deduced amino acid sequence of the cytochrome subunit in Rsp. molischianum contains significant deletions at the attachment site to the M subunit compared with that of Rhodopseudomonas viridis. This suggests that the interaction between the cytochrome subunit and the LM core in Rsp. molischianum and Rvi. gelatinosus is different from that in Rps. viridis. Phylogenetic analysis of the light-harvesting proteins indicated that the LH1 and subunits of Rsp. molischianum are included in the lineage of LH1 polypeptides of the purple bacteria, while the LH2 and subunits are positioned apart from LH2 polypeptides of the other purple bacteria together with those of Chromatium vinosum. Based on these phylogenetic analyses, the classification of the light-harvesting proteins in purple bacteria is discussed.     

The PucC protein of Rhodobacter capsulatus mitigates an inhibitory effect of light-harvesting 2 α and β proteins on light-harvesting complex 1

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.     

Gibberellin metabolism in excised lettuce hypocotyls: Evidence for the formation of gibberellin A1 glucosyl conjugates

The properties of the water-soluble metabolites of [³H]gibberellin A₁ ([³H]GA₁) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA₁ or GA₃. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated ¹⁴C from glucose and ³H from GA₁. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [¹⁴C]glucose together with a second uncharacterised component. Feeding with [³H]GA₁ methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA₁-glucosyl ester (LH 1) and GA₁-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA₃-glucosyl ester - LH2 GA₁-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography     

Detection of an open reading frame related to the CMS-associated urf-s in fertile Petunia lines and species and in other fertile Solanaceae species

Summary In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 3511-E, 1991 series     

Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants. This revised version was published online in August 2006 with corrections to the Cover Date.     

多粘类芽胞杆菌KM2501-1杀南方根结线虫活性产物研究

【目的】南方根结线虫(Meloidogyne incognita)是一种危害严重的土传性植物病原线虫,给农业生产造成了巨大的经济损失,前期研究发现多粘类芽胞杆菌(Panebacillus polymyxa) KM2501-1具有很好的温室防治南方根结线虫效果,且可产生多种挥发性杀线虫活性物质,但对其非挥发性产物是否有杀线虫活性没有研究。本研究拟进一步分离鉴定其产生的杀线虫活性代谢产物,发掘新的杀线虫药物。【方法】对菌株KM2501-1进行液体发酵并离心收集发酵上清液,通过硅胶柱层析、高效液相色谱分离等方法得到高纯度的杀线虫活性物质,并通过液相色谱质谱联用分析、核磁共振等技术鉴定杀线虫活性物质的结构。【结果】生物活性检测显示,多粘类芽胞杆菌KM2501-1发酵上清液具有较强的南方根结线虫触杀活性,并能有效抑制南方根结线虫卵孵化,体外杀线虫效率高达87.66%,抑制卵孵化效率达92.26%。结构鉴定结果显示多粘类芽胞杆菌产生的杀线虫活性物质为环二肽类物质cyclo (Pro-Phe),800 mg/L的cyclo(Pro-Phe)杀线虫效率达84.75%。进一步的显微观测结果表明,与对照组相比,活性物质cyclo(Pro-Phe)处理后的根结线虫肠道组织紊乱、结构发生破坏。【结论】多粘类芽胞杆菌KM2501-1产生的cyclo (Pro-Phe)是一个具有杀线虫新功能的活性物质,其可能通过破坏线虫肠道杀死线虫。