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Unlabelled: ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070±150kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance., Statement of Significance: Extracellular matrix (ECM)-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. One such ECM-based material, cartilage ECM, has recently shown potential to be chondroinductive; however, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. Therefore, this work is significant because we were the first to create hydrogels derived entirely from cartilage ECM that had mechanical properties similar to that of native cartilage until hydrogel failure. Furthermore, these hydrogels had a compressive modulus of 1070±150kPa, they were chondroinductive, and they supported extensive matrix synthesis. In the current study, we have shown that these new hydrogels may prove to be a promising biomaterial for cartilage tissue engineering applications., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6 weeks with and without exposure to human transforming growth factor β3 (TGF-β3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6 weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-β3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII(-) mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation.

Cells of Rhv. sulfidophilum were grown under different conditions in the presence of 32P-phosphate and the corresponding H and L membrane fractions obtained and fractionated by SDS-PAGE. Both membranes showed almost identical polypeptide composition. The bacteriochlorophyll (Bchl) specific content in H was always lower that in L. As described before, oxygen did not regulate gene expression. Under high light, an almost two- to threefold decrease of the cellular specific Bchl content was observed. Pulse and chase experiments showed that transitions from aerobiosis to light-anaerobiosis did not quantitatively affect the Bchl content of the membranes, although a turnover of the 32P-phosphate and 35S-methionine was observed. LHI beta was the only polypeptidic subunit of the Bchl-binding polypeptides that was phosphorylated in vivo, and phosphotyrosine was the only phosphorylated amino acid detectable. The phosphorylated LHI beta was determined to be insoluble in the organic solvent mixture of (vol/vol) 1:1 chloroform-methanol containing ammonium acetate (0.1 m final concentration). Treatment with a chaotropic agent such as Na2CO3 solubilized the phosphorylated LHI beta, indicating that part of this posttranslationally modified polypeptide was not inserted in a transmembrane position. These results were used to speculate about the regulatory properties of this posttranslational modification of LHI beta on membrane differentiation.

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.

The radC gene, whose product plays a role in the prokaryotic repair of DNA damage after UV and X-ray irradiation, was cloned and sequenced from the phototrophic bacterium Rhodobacter capsulatus B10. The gene codes for a protein of 214 amino acids with a molecular mass of 23,792 Da. The deduced amino acid sequence showed significant homology with the RadC proteins of Escherichia coli, Bacillus subtilis and Haemophilus influenzae. Northern blot analysis indicated that under both chemotrophic and phototrophic growth conditions the radC gene was relatively highly expressed and was induced about five-fold after UV-irradiation. Primer extension analysis revealed that transcription was initiated from the same position before and after UV treatment. Mutants (radC negative) have a low survival rate and a slower growth rate than the wild type.

In the present work we report the high-level expression of foreign genes encoding the light-harvesting (LHII) membrane-spanning polypeptides in photosynthetic bacteria. To do this we first constructed three deletion strains of Rhodovulum (Rhv.) sulfidophilum in which all or part of the puc operon, encoding the peripheral light-harvesting proteins, is missing. To investigate the heterologous expression of the light-harvesting polypeptides from Rb. capsulatus in Rhv. sulfidophilum and vice versa we have reintroduced functional foreign LH genes into these and equivalent strains of Rhodobacter (Rb.) capsulatus. In some cases very high levels of expression were obtained (85%) of those observed in the wild type), while in other cases much lower expression was observed; possible reasons for these differences are discussed. The heterologously expressed proteins were shown to contain normal pigment-binding sites and to be normally and functionally integrated within the host photosynthetic apparatus. The results indicate that heterologous proteins are able to assemble properly and enter into the same protein-protein interactions as their analogs originally present in the host strain.

The genes somB and somA (Synechococcus outer membrane), lying in tandem organization in the genome of Synechococcus PCC 6301, encode two porins in the outer membrane of this unicellular cyanobacterium. Northern blot and primer extension experiments revealed that somA and somB are not comprising an operon, as each gene encodes a transcript of 1.7 kb length and has a distinct transcriptional start site. The deduced SomA and SomB protein sequences include typical N-terminal signal peptides and reveal 60% homology (50% identical residues) to each other as well as significant homology to six protein sequences deduced from open reading frames sequenced in the genome of the unicellular cyanobacterium Synechocystis PCC 6803. Furthermore, SomA possesses an overall identity of 97% to the functionally not yet characterized outer-membrane protein SomA from the closely related cyanobacterial strain Synechococcus PCC 7942. Analyses performed on the sequences suggest that SomA and SomB form 14- or 16-stranded porin-like beta-barrels. Moreover, all sequences share an N-terminal motif with significant homology to 'S-layer homology' domains, which might form a periplasmic extension. SomA and SomB therefore may, in addition to their porin function, act as linkers connecting the outer membrane with the peptidoglycan layer.

Two major proteins, A and B, were isolated and purified from outer membranes of the unicellular cyanobacterium Synechococcus PCC 6301 by gel filtration, anion-exchange chromatography, and preparative SDS-PAGE. Protein A revealed a single-channel conductance of 0.4 nanoSiemens (nS) in 1 M KCl, whereas preparations containing both proteins showed two different conductance maxima of 0.4 and 0.9 nS, suggesting that B also forms pores. The apparent molecular mass of the two closely migrating proteins was determined as 52 kDa, whereas native porin extracts revealed a relative molecular mass of ca. 140 kDa, indicating trimeric pore-forming units. Partial sequences of both proteins were obtained by N-terminal sequencing of tryptic peptides, and the C-terminal amino acid sequences were derived from the complete proteins. These sequences were aligned to protein sequences available in the databases. The results are discussed.

In Rhodobacter capsulatus (Rbc), the participation of DnaK in the synthesis of light harvesting antenna complex I (LHI) has been recently inferred from the finding that the amount of LHI alpha- and beta-polypeptides synthesized in an in vitro translation system was strongly reduced when DnaK was depleted. In the present work, a DnaK protein was isolated from Rbc and biochemically characterized. The N-terminus of the protein was sequenced and a corresponding oligo was used as probe in order to clone the gene coding for DnaK. The dnaK gene was located in an operon (dnaKJ) with two open reading frames, which code for DnaK and DnaJ, respectively. A promoter element corresponding to the consensus sequence of the atypical heat shock (HS) promoter of several alpha-purple proteobacteria was identified. Northern blot analysis indicated that dnaK and dnaJ belong to the same transcriptional unit; there were two transcripts, one comprising both the dnaK and dnaJ genes and a second with only dnaK. Primer extension analysis revealed that under both chemotrophic and phototrophic conditions transcription was initiated from the same position before and after HS. The promoter activity was studied under different growth conditions with a dnaK-lacZ fusion under the control of the dnaKJ promoter. The present work opens up the possibility to study the specific role of DnaK in the assembly of photosynthetic apparatus proteins.

Rhodovulum (Rhv.) sulfidophilum, unlike other nonsulfur purple bacteria, is able to synthesize the peripheral antenna complex even under fully aerobic conditions in the dark. We have obtained strong evidence that Rhv. sulfidophilum encodes only one copy of the puc operon, comprising pucB, pucA and pucC. pucB and pucA encode the beta- and alpha-polypeptides. The third ORF (pucC), downstream of pucA, has a strong homology to pucC of Rhodobacter (Rb.) capsulatus. Deletion mutation analysis indicated that the requirement for the pucC gene product for LH II expression was less strict than in Rb. capsulatus. Comparison of the deduced alpha and beta polypeptide sequences with the directly determined primary structure revealed a C-terminal processing of the alpha-subunit. Primer extension analysis showed that the pucBAC is transcribed from a sigma70-type promoter 130 bases upstream of the translational start of pucB. Transcriptional expression of the pucBAC operon in Rhv. sulfidophilum is higher, the lower the light intensity is, and is not reduced to a ground-level by the presence of oxygen. Based on lacZ fusions the relative promoter activities were, for dark aerobic:dark semiaerobic:low light anaerobic:medium light anaerobic:high light anaerobic, 5.5:7.0:2.0:1.0:0.78. Still unidentified cis-regulatory elements or binding sites of trans-regulatory elements are apparently localized in two distinct upstream regions. Furthermore, comparison of the promoter region of the Rhv. sulfidophilum pucBAC with the promoter regions of puc operons in related species showed distinct differences in the regulatory elements. The significance of these results with respect to the regulation of transcription and the oxygen-independent synthesis of LH II from Rhv. sulfidophilum is discussed.

The primary and atomic structures of the porin protein from Rhodobacter (Rb.) capsulatus strain 37b4 were determined several years ago by peptide sequencing and X-ray crystallography. In this work the gene encoding this porin (named porCa) was cloned and sequenced. The porin open reading frame encodes 320 amino acids-a mature protein of 300 residues (molecular mass 31 552 kDa) and a presequence of 20 amino acids. Our deduced amino-acid sequence was directly confirmed by purifying the porin protein from the same bacterial strain and sequencing the amino terminus as well as several peptides derived from trypsin digestion. However, comparison of this deduced amino-acid sequence with the published primary structure of this porin, nominally from the same strain (but cultivated for ca. 30 years in a different laboratory) reveals seven differences in the amino-acid sequence at the following positions in the mature protein (published/present): 59 (Gly/Ala), 123 (Tyr/Asn), 135 Ser/Thr), 189 (Ile/Val), 196 (Asn/His), 231 (Ala/Thr) and 238 (Ser/deleted). Surprisingly, analysis of the positioning of these mutations revealed that they are located exclusively on transmembrane strands, with two of them deeply buried within the structure. These mutations may in fact have only marginal influence on porin structure and function. Northern blot analysis revealed that porCa encodes an RNA transcript of 1070 nucleotides. No differential response in the abundance or size of this mRNA was seen upon growth under phototrophic/anaerobic vs. chemotrophic/aerobic conditions, under high or low osmotic pressure. Primer extension experiments revealed a transcription start site 73 bases upstream from the ATG translation start, juxtaposed to the identified putative promoter region. Fusion of lacZ with this putative promoter region (using a 288-bp upstream region) revealed similar promoter activity in beta-galactosidase assays under both physiological conditions tested, again suggesting that this gene is constitutively expressed. The molecular genetic characterization described in this work opens the way for structure-function studies by site-directed mutagenesis.

The pore-forming outer-membrane protein from Rhodobacter (R.) capsulatus (wild-type B10 strain) was isolated and purified under non-denaturing conditions. The monomer unit of the isolated porin has a molecular mass of about 28 kDa, as judged by SDS-PAGE, whereas the native protein migrates at 75 kDa. This suggests that the native porin from R. capsulatus B10 exists in a trimeric form. The N-terminal amino acid sequence was used to design an oligonucleotide which was utilised to screen a pBluescript library containing EcoRI fragments of R. capsulatus B10 DNA. A 5.3-kb DNA fragment, which included the entire structural porin gene (named porCa) and its flanking regions, was identified. A 945-bp open reading frame, coding for a mature protein of 295 amino acid residues (molecular mass 30,586 Da) plus a presequence of 20 amino acids, was found. The directly determined sequence of the amino-terminus and of four tryptic peptides of the purified porin matched perfectly with the deduced amino acid sequence. Northern blot analysis showed that the porin gene encodes an RNA transcript of 1050 nucleotides. In addition, there is no differential response in terms of either the size or abundance of the mRNA under different environmental conditions. Primer extension experiments confirmed a putative promoter upstream of the porin gene; and localised the RNA transcription start site 73 bp upstream of the ATG start codon, which is close to the putative promoter (-10/-35). As shown using a plasmid-borne porin-lacZ gene fusion, [in R. capsulatus] expression of the lacZ gene under the control of the porCa promoter is not regulated under the two different environmental conditions tested. The promoter of the porin gene was localised within 305 bp upstream of the ATG start codon. A model of the 3-D structure of porin B10 was deduced by comparative modelling with the R. capsulatus 37b4 and Rhodopseudomonas blastica porin crystallographic structures using the ProMod program on the Swiss-Model protein modelling e-mail Server. Analysis of the B10 sequence and comparison of a model of the B10 porin structure with the crystallographic structure of porin from the capsuleless strain 37B4 has revealed some important differences at the level of the protein surface, the pore and the putative ligand binding site.

The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a -35/-10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45-75 min after shifting from 28 degrees C to 39 degrees C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.

Porin was isolated from Rhodobacter (Rb.) capsulatus wild type B10, as well as from Rb. capsulatus 37b4 as a control. The porin from Rb. capsulatus B10 shows significant differences to that of Rb. capsulatus 37b4 in N-terminal sequence, amino acid composition and molecular mass. The apparent molecular mass of the purified porin from Rb. capsulatus B10 is about 28 kDa by SDS-PAGE, whereas the native porin trimer migrates at 75 kDa. For Rb. capsulatus 37b4 the molecular mass of the momomer is about 30 kDa and for the trimer about 76 kDa. These differences may be related to the morphological differences between the two wild type strains. Rb. capsulatus B10 has an extracellular capsule whereas Rb. capsulatus 37b4 is capsuleless. The native proteins from both wild types showed similar single channel conductance measurements in black lipid membranes. The B10 porin has been crystallised using the detergent beta-d-octyl-gluco-pyranoside. The crystals diffracted to 3 A and belong to the orthorhombic space-group P212121. The crystal packing could be elucidated by molecular replacement.

The expression of the Rhodobacter capsulatus catalase-peroxidase (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene. The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions. The promoter was localized within a 69-bp upstream DNA region. The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis. Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.

The enzymic activity of phytoene desaturase in Narcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox-active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone-rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox-active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43-kDa NAD(P)H:quinone oxidoreductase, which is organized as a homodimer (23 +/- 3 kDa/subunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23-kDa protein of the oxygen-evolving complex of photosystem II, but no information about the N-terminal organization of the oxidoreductase could be obtained. During flower development, the steady-state concentration of the corresponding mRNA is up-regulated, indicating a specific function of the gene product in chlorophyll-free chromoplasts.

We demonstrate that the B800-830 spectral form of the B800-850 peripheral light-harvesting complex of Rhodobacter sulphidophilus, which is formed at low ionic strengths in the presence of the zwitterionic detergent LDAO, results from a local modification of the bacteriochlorophyll binding site and not the dissociation of the complex. This perturbation does not result in significant changes to the interactions between the pigments as studied by circular dichroism or fluorescence spectroscopy; however, modifications in the pigment binding sites are inferred from changes in the preresonance Raman spectrum. Specifically, an alteration of the hydrogen bonding of the 2-acetyl group of at least one of the bacteriochlorophyll groups that make up the 850 nm absorbing pair is observed. This implies an alteration in the conformation of the C-terminal domain of the alpha-polypeptide, in which are located the two tyrosyl residues that are believed to act as H-bond donors to these groups, induced by the protein-bound detergent in the absence of bound cations. We suggest that the ability of this complex to form an 800-830 complex is linked to the presence of an aspartyl residue immediately upstream of the tyrosyl residues. This study therefore provides a further illustration of the importance of hydrogen bonds to the 2-acetyl group of the bacteriochlorophyll in the determination of its spectral properties; furthermore, we provide a description of a conformational change that is able to modulate chromophore binding in these complexes.

The peripheral light-harvesting bacteriochlorophyll-carotenoid-protein complex B800-850 (LHII) has been isolated from membranes of semi-aerobic dark-grown cells of Rhodobacter sulfidophilus strain W4. A reversed-phase HPLC system resolved one beta- and one alpha-polypeptide in the ratio 1:1. The material obtained was of high purity and suitable for direct microsequence analysis. The primary structures of the beta- and alpha-polypeptides have been determined. The beta-polypeptide consists of 51 amino acid residues, yielding a molecular mass of 5512 Da and having 64.7% hydrophobicity. The alpha-polypeptide consists of 52 amino acid residues, with a calculated molecular mass of 5661 Da and 75% hydrophobicity. The significance of uncommon structure motives with respect to the unusual spectroscopic characteristics of this light-harvesting complex is discussed.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

Pore-forming protein (porin) was isolated from N,N-dimethyl-dodecylaminoxid (LDAO)-extracted outer membranes of Synechococcus PCC 6301 and purified by ion exchange chromatography on DEAE-Sephacel column. The apparent molecular mass on SDS-PAGE was determined to be about 52,000. The native porin was reconstituted into black lipid bilayer membranes and showed a single-channel conductance of 5.5 nS in 1 M KCl. The porin was found to be N-terminally blocked. The C-terminal amino acid sequence was identified as Phe-Thr-Phe. Amino acid analysis suggested that the porin protein consists of about 420 amino acid residues, yielding a polarity of 43.6% and a molecular mass of 45,000 in contrast to the mobility on SDS-PAGE.

The genome of Rhodopseudomonas palustris contains five antenna gene clusters, alpha beta a, alpha beta b, alpha beta c, alpha beta d and alpha beta e, which encode the light-harvesting peripheral antenna complex II polypeptides. The isolation and characterisation of the gene which encodes the alpha e and beta e polypeptides are reported. The primary structure of the beta e polypeptide is identical to that of beta b whilst the structure of alpha e is different from the other alpha subunits so far characterised. All five of the gene clusters were transcribed under high-light conditions while under low-light conditions only three were transcribed (alpha beta b, alpha beta d and alpha beta e). Furthermore, Northern-blot analysis showed that the gene clusters encode RNA transcripts of either 500 or 650 nucleotides. Individual members of the gene family showed a differential response in terms of the regulation of abundance of mRNA upon growth under either high-light or low-light intensities. Possible promoter sequences and operator sites upstream of the alpha beta b, alpha beta d and alpha beta e genes were located. Furthermore using puc-lacZ fusions in trans in R. palustris, we were able to examine the positions of the promoter of the gene clusters. The significance of these observations with respect to the regulation, organization and role of the peripheral antenna is discussed.

The gene encoding catalase-peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7-kb SacI-HindIII fragment, containing the catalase-peroxidase gene (cpeA) and its flanking regions were determined. A 1728-bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed. A Shine-Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase-peroxidase of R. capsulatus B10. The amino acid sequence of R. capsulatus catalase-peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase-peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase-peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.

The transcription factors LF-A1 and LF-B1 are required for the cell-specific expression of the human alpha 1-antitrypsin gene in hepatocytes. We report here the purification and preliminary characterization of LF-A1. This protein, purified to homogeneity from calf liver nuclei by site-specific DNA affinity chromatography and reverse-phase HPLC, has a molecular mass of 40 kDa. Binding sites of LF-A1 are present in the promoter regions of several genes expressed in the liver (alpha 1-antitrypsin, apolipoproteins A1, B1, A4 and pyruvate kinase). Interestingly, the binding site of LF-A1 is bipartite and consists of two short sequence motifs (consensus: TGGACT/CT/C and TGGCCC) separated by a variable 'spacer' region. Insertion or deletion of 1-4 nucleotides in the 'spacer' region of the site in the alpha 1-antitrypsin promoter does not abolish DNA binding.

A new procedure for isolation, purification and identification of the three polypeptides of the membrane-bound light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata has been developed. The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion-exchange chromatography. The three major polypeptides isolated by this two-step chromatography were found to be homogenous and identical with the three polypeptides of the light-harvesting complex II, as judged by amino acid analysis and N-terminal sequence determination. Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800-850 complex studied by the same criteria.

A reverse-phase HPLC System for isolation of the water insoluble alpha- and beta-polypeptides of the light-harvesting complex II (LH II) of Rhodopseudomonas (Rps.) palustris without employment of any detergent was developed. The material obtained was of high purity and suitable for direct microsequence analysis. Chromatographic analysis could resolve at least two major beta-polypeptides, beta a and beta b, two major alpha-polypeptides, alpha a and alpha b, and two additional minor polypeptides. N-terminal amino acid sequencing shows that the resolved peaks correspond to different polypeptide species and that the minor species have an N-terminal sequence identical to that of the alpha b polypeptide. An oligonucleotide derived from the amino terminal sequence of the alpha a polypeptide was utilized to screen a genomic library from Rps.palustris. Several independent clones have been characterized by Southern blot and nucleotide sequence analysis. We show that Rps.palustris contains at least four different clusters of beta and alpha genes. Two clones contain sequences potentially coding for beta a-alpha a and beta b-alpha b polypeptides; and two additional clones potentially coding for beta and alpha peptides which we named beta c-alpha c and beta d-alpha d, which did not correspond to the major purified polypeptides. In addition to the protein chemistry data, the conservation at the amino acid level and the presence of canonical ribosomal binding sites upstream of each of the identified genes strongly suggest that all four coding regions are expressed.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.

[14C]penicillin binding experiments and membrane analysis were carried out with cell envelope preparations from Escherichia coli and Proteus mirabilis. After incubation with [14C]penicillin G labeled free lipoprotein could be identified. The analysis of the isolated lipoprotein by SDS polyacrylamide gel electrophoresis indicates that there is only one protein with an apparent molecular weight of 7000. The amino acid composition of isolated labeled free lipoprotein from E. coli was identical to the lipoprotein already found in E. coli. It is a point of interest that the amino acid composition of the isolated labeled free lipoprotein from P. mirabilis D 52 differs from that found in other mutants of this strain. The free form of lipoprotein from P. mirabilis D 52 is composed of 61 amino acids and has glycine, phenylalanine and proline as specific components.

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.