The LHIalpha and LHIIalpha complexes in association with phospholipids are able to be inserted in heavy membranes of Rhodobacter capsulatus B10.

We show in this paper that a complex constituted by phospholipids and LHI and LHII alpha polypeptides was inserted in a heavy membrane fraction in a nonextractable form, indicating a transmembrane localization. The best accepting membranes originated from aerobically grown cells. Addition of ATP during the insertion inhibited this reaction 25 to 30% in heavy membranes isolated from aerobically grown cells (HMaer) and a higher inhibition (60 to 65%) was detected when using heavy membranes isolated from photosynthetically grown cells (HMpho). Purification by gel filtration of a crude Na2CO3 extract yielded three phosphate-labeled fractions. Two of them contained protein and phospholipids in a stable association. However, only fractions containing phosphatidylethanolamine were shown to be reconstituted. The third radioactive fraction contained labeled ATP and protein, but no phospholipids and could not be reassociated to the heavy membranes of any origin. A model for the insertion of the LH polypeptides is presented in which the recently synthesized polypeptides are phosphorylated and become associated to anionic phospholipids. The interaction of this complex to the membrane spontaneously leads to stable insertion.

Molecular analysis and identification of the radC gene from the phototrophic bacterium Rhodobacter capsulatus B10.

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.

Heterologous expression of genes encoding bacterial light-harvesting complex II in Rhodobacter capsulatus and Rhodovulum sulfidophilum.

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.

Gene cloning and regulation of gene expression of the puc operon from Rhodovulum sulfidophilum.

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.

Isolation and complete amino acid sequence of the beta- and alpha-polypeptides from the peripheral light-harvesting pigment-protein complex II of Rhodobacter sulfidophilus.

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.

Cloning of a new antenna gene cluster and expression analysis of the antenna gene family of Rhodopseudomonas palustris.

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.

Anion channel forming activity from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense.

The plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense secretes an anion channel forming activity (CFA) into the culture field. The CFA inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase of the bilayer chamber. The channels formed are highly anion selective. The conductance decreases for larger anions (Cl- greater than SCN- greater than SO2-(4] and is practically zero for gluconate. The channels show a unique voltage dependence: (i) The single-channel conductance increases linearly with voltage up to 200 mV saturating at 250 mV with 25 +/- 1 pS (300 mM KCl). The channel is closed at negative voltage relative to the side of insertion (diode-type I-V curve). (ii) The average number of open channels also increases with voltage. The Poisson distribution of channel numbers indicates independent opening of the channels. Channel activity can be abolished by protease treatment of the planar bilayer. The channels can be blocked by indanyloxyacetic acid (IAA-94) and by pH greater than 10. The CFA was purified yielding one major band on the SDS gel with a relative molecular mass of 65,000. The putative involvement of the CFA in the toxicity of this plant pathogen is discussed and compared to other toxins like colicins and to the diptheria toxin group.