Functional expression of nitrogenase-like protochlorophyllide reductase from Rhodobacter capsulatus in Escherichia coli

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction of protochlorophyllide in chlorophyll and bacteriochlorophyll biosynthesis. DPOR consists of two components, L-protein and NB-protein, which are structurally related to nitrogenase Fe-protein and MoFe-protein, respectively. Neither Fe-protein nor MoFe-protein is expressed as an active form in Escherichia coli due to the requirement of many Nif proteins for the assembly of the metallocenter and the maturation specific for diazotrophs. Here we report the functional expression of DPOR components from Rhodobacter capsulatus in Escherichia coli. Two overexpression plasmids for L-protein and NB-protein were constructed. L-protein and NB-protein purified from E. coli showed spectroscopic properties similar to those purified from R. capsulatus. L-protein and NB-protein activities were evaluated using a crude extract of E. coli overexpressing NB-protein and L-protein, respectively. Specific activities of the purified L-protein and NB-protein were 219+/-38 and 52.8+/-5.5 nmolChlorophyllide min(-1) mg(-1), respectively, which were even higher than those of L-protein and NB-protein purified from R. capsulatus. These E. coli strains provide a promising system for structural and kinetic analyses of the nitrogenase-like enzymes.     

Conservation and variation between Rhodobacter capsulatus and Escherichia coli Tat systems

The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a complete Tat translocon that Tat systems are largely, but not fully, interchangeable even between different classes of proteobacteria. The Tat apparatus from the alpha-proteobacterium Rhodobacter capsulatus was transferred to a Tat-deficient Escherichia coli strain, which is a gamma-proteobacterium. Similar to that of E. coli, the R. capsulatus Tat system consists of three components, rc-TatA, rc-TatB, and rc-TatC. A fourth gene (rc-tatF) is present in the rc-tatABCF operon which has no apparent relevance for translocation. The translational starts of rc-tatC and rc-tatF overlap in four nucleotides (ATGA) with the preceding tat genes, pointing to efficient translational coupling of rc-tatB, rc-tatC, and rc-tatF. We show by a variety of physiological and biochemical assays that the R. capsulatus Tat system functionally targets the E. coli Tat substrates TorA, AmiA, AmiC, and formate dehydrogenase. Even a Tat substrate from a third organism is accepted, demonstrating that usually Tat systems and Tat substrates from different proteobacteria are compatible with each other. Only one exceptional Tat substrate of E. coli, a membrane-anchored dimethyl sulfoxide (DMSO) reductase, was not targeted by the R. capsulatus Tat system, resulting in a DMSO respiration deficiency. Although the general features of Tat substrates and translocons are similar between species, the data indicate that details in the targeting pathways can vary considerably.     

Primary structure of porin from Rhodobacter capsulatus.

The primary structure of the integral membrane protein porin from the purple bacterium Rhodobacter capsulatus was determined. The protein was cleaved with trypsin, CNBr and Asp-N protease. The peptides were isolated, sequenced and aligned to a total length of 301 residues with an Mr of 31,536. The low isoelectric point of 3.9 is confirmed by the high excess of 34 Asp and 17 Glu (16.9%) over 10 Lys, 7 Arg and 2 His (6.3%). Overall sequence similarity to other porins is not evident when using sequence alignment programs. However, a partial relationship to Neisseria porins seems to exist. The established sequence has been used as the basis for a three-dimensional structure determination by X-ray diffraction at 0.18-nm resolution. The arrangement of the sequence in the 16-stranded beta-barrel of porin is given. Some sequence-structure correlations are discussed.     

RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.     

The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase.

The essential gene efg, which complements ammonia-dependent growth (adgA) mutations in Rhodobacter capsulatus and is located at 38.1 min on the Escherichia coli chromosome, was found to code for NH3-dependent NAD synthetase. Crude extracts from a strain which overproduces the efg gene product contained up to 400 times more activity than crude extracts from the control strain, and the purified Efg protein possessed-NH3-dependent NAD synthetase activity. Glutamine-dependent NAD synthetase activity was found in crude extracts of E. coli but not in the purified enzyme, suggesting that it may be catalyzed by an additional subunit. An R. capsulatus strain carrying an adgA mutation was found to be deficient in NAD synthetase activity, and activity was restored by complementation with the E. coli gene. In accordance with the nomenclature proposed for Salmonella typhimurium (K. T. Hughes, B. M. Olivera, and J. R. Roth, J. Bacteriol. 170:2113-2120, 1988), the efg and adgA genes should now be designated nadE.     

Nucleotide transport in Rhodobacter capsulatus.

Cytoplasmic membrane vesicles isolated from the gram-negative photosynthetic bacterium Rhodobacter capsulatus catalyzed the transport of nucleotides. No transport occurred in the intact bacteria unless they were pretreated with EDTA. The transport rate was measured by incorporation of radioactive phosphate into externally added ADP or by incorporation of nonradioactive phosphate into added labeled ADP. The catalytic activities which utilized the added ADP were photosynthetic ATP synthesis, Pi-ADP exchange, and adenylate kinase. These activities were shown to occur on the cytoplasmic side of the internal membrane. The products were found in the outer medium. The rate of nucleotide transport across the membranes was comparable to the rate of photophosphorylation. These results indicated that nucleotides can be transported across the cytoplasmic membrane but not across the outer membrane of the native R. capsulatus cell. Therefore, by analogy to the mitochondrial ATP-ADP translocator, the exchange might function as an energy transfer system to the periplasm of these bacteria.     

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.     

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.     

Physiological functions of hydroperoxidases in Rhodobacter capsulatus.

Rhodobacter capsulatus J1 has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H2O2 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H2O2 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.     

Thiolases of Escherichia coli: purification and chain length specificities.

The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated. A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation. The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis. The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates. In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A.     

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.