Crystallization and preliminary X-ray analysis of porin from Rhodobacter capsulatus

Porin monomers of the phototrophic bacterium Rhodobacter capsulatus were purified. Crystals were obtained from a solution of porin solubilized with the detergent octyltetraoxyethylene within 5 days using the vapor phase equilibration technique. The crystals were rhombohedral with an edge length of 0.4 mm. The space group was trigonal R3 with unit cell constants of a = b = 95 A, c = 147 A. Reflexions were observed to 3.2 A.     

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

Abstract The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene ( bfr ) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli . The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.     

Cloning and characterization of the ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.     

Export of porin to the outer membrane of the phototrophic bacterium Rhodobacter capsulatus 37B4

Export of porin to the outer membrane of the phototrophic purple bacterium Rhodobacter capsulatus was studied with the use of the uncoupler of the electron transport chain, carbonylcyanide-m-chlorophenylhydrazone (CCCP). The agent reversibly blocked the transport of porin across the cytoplasmic membrane. By means of radioactive labeling and immunoprecipitation, porin was found to occur in two forms: (i) the exported form that was extractable from the outer membrane without disrupting the cells, and (ii) a pre-form with a slightly higher apparent molecular mass which accumulated in the cells during the block of the export process. Proteolysis studies revealed that the preform was highly sensitive to added proteases, whereas the exported form was resistant.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - OMP outer membrane porin; pre-OMP, form of outer membrane porin before export - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Effects of cadmium stress on growth, morphology, and protein expression in Rhodobacter capsulatus B10

The effects of cadmium stress on growth, morphology, and protein expression were investigated in Rhodobacter capsulatus B10 using two-dimensional polyacrylamide gel electrophoresis and a scanning electron microscope with an energy dispersive X-ray spectrometer. The bacterium grew in the presence of 150 microM CdCl2 and highly induced heat-shock proteins (GroEL and Dnak), S-adenosylmethionine synthetase, ribosomal protein S1, aspartate aminotransferase, and phosphoglycerate kinase. Interestingly, the ribosomal protein S1 was proportionally expressed as the amount of cadmium in the medium, suggesting that S1 may be required for the repair of cadmium-mediated cellular damage. On the other hand, we identified five cadmium-binding proteins: 2-methylcitrate dehydratase, phosphate periplasmic binding protein, inosine-5'-monophosphate dehydrogenase/guanosine-5'-monophosphate reductase, inositol monophosphatase, and lytic murein transglycosylase. The cadmium-treated cells had a filamentous structure and contained less phosphorous than the untreated cells. We propose that these characteristics of the cadmium-treated cells may be due to the inactivation of the phosphate periplasmic binding protein and lytic murein transglycosylase by cadmium.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34?kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R.?capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34?kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R.?capsulatus cultures.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.     

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.     

Cloning and overexpression of the Rhodobacter capsulatus hemH gene.

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.     

Identification and analysis of the rnc gene for RNase III in Rhodobacter capsulatus.

The large subunit ribosomal RNA of the purple bacterium Rhodobacter capsulatus shows fragmentation into pieces of 14 and 16S, both fragments forming the functional equivalent of intact 23S rRNA. An RNA-processing step removes an extra stem-loop structure from the 23S rRNA [Kordes, E., Jock, S., Fritsch, J., Bosch, F. and Klug, G. (1994) J. Bacteriol., 176, 1121-1127]. Taking advantage of the fragmentation deficient mutant strain Fm65, we used genetic complementation to find the mutated gene responsible for this aberration. It was identified as the Rhodobacter homologue to mc from Escherichia coli encoding endoribonuclease III (RNase III). The predicted protein has 226 amino acids with a molecular weight of 25.5 kDa. It shares high homology with other known RNase III enzymes over the full length. In particular it shows the double-stranded RNA-binding domain (dsRBD) motif essential for binding of dsRNA substrates. The Fm65 mutant has a frame shift mutation resulting in complete loss of the dsRBD rendering the enzyme inactive. The cloned Rhodobacter enzyme can substitute RNase III activity in an RNase III deficient E. coli strain. Contrary to E. coli, the Rhodobacter mc is in one operon together with the lep gene encoding the leader peptidase.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.