Green Sulfur Bacterium Chlorobium Research Articles

Molecular Contacts for Chlorosome Envelope Proteins Revealed by Cross-Linking Studies with Chlorosomes fromChlorobium tepidum

Chlorosomes are unique light-harvesting antennae found in two phyla of green bacteria: Chlorobi and Chloroflexi. In the green sulfur bacterium Chlorobium tepidum, 10 proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) exist in the chlorosome envelope. Chlorosomes from the wild type and mutants lacking a single chlorosome protein were cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and analyzed by gel electrophoresis. Similar cross-linking products were observed when the time and temperature were varied or when EDC was replaced with glutaraldehyde. Specific interactions between chlorosome proteins in cross-linked products were identified by immunoblotting with polyclonal antibodies raised against recombinant chlorosome proteins. We confirmed these interactions by demonstrating that these products were missing in appropriate mutants. Confirming the location of CsmA in the paracrystalline baseplate, cross-linking showed that CsmA forms dimers, trimers, and homomultimers as large as dodecamers and that CsmA directly interacts with the Fenna-Matthews-Olson protein. Cross-linking further suggests that the precursor form of CsmA is inserted near the edges of the baseplate, where CsmA and pre-CsmA interact with CsmB and CsmF. Several chlorosome proteins, including CsmA, CsmC, CsmD, CsmH, CsmI, CsmJ, and CsmX, were shown to exist as homomultimers in the chlorosome envelope. On the basis of the structural information obtained from these cross-linking experiments, a model for the locations and interactions of the proteins of the chlorosome envelope is proposed.

Soluble cytochrome c-554, CycA, is not essential for photosynthetic electron transfer in Chlorobium tepidum

We constructed a mutant lacking soluble cytochrome c-554 (CycA) by disruption of the cycA gene in the green sulfur bacterium Chlorobium tepidum. The mutant grew phototrophically with a growth rate slower than that of the wild type, suggesting that CycA is not essential for photosynthetic electron transfer even though CycA is known to work as an electron donor to the reaction center. The re-reduction of photo-oxidized cytochrome c z by quinol oxidoreductase was inhibited almost completely by the addition of stigmatellin in the mutant cells. This result indicates that, in the mutant cells, the linear electron transfer can occur from the quinol oxidoreductase to cytochrome c z, and to reaction center P840 with no participation of CycA.

2P349 Fast electron donation from CycA to the homodimeric type-I reaction center of the green sulfur bacterium Chlorobium tepidum(43. Photosynthesis,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P349 Fast electron donation from CycA to the homodimeric type-I reaction center of the green sulfur bacterium Chlorobium tepidum(43. Photosynthesis,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Assembly of extramembranous light-harvesting complexes of green sulfur photosynthetic bacterium Chlorobium tepidum on glass surface by electrostatic layer-by-layer adsorption

Extramembranous and peripheral light-harvesting complexes (chlorosomes) of green sulfur photosynthetic bacterium Chlorobium tepidum were electrostatically immobilized on a pyrex glass plate with the help of cationic polylysines. Absorbance of both Soret and Q y bands of chlorosomes were linearly increased by repeated manipulation of layer-by-layer adsorption. Spectral features of chlorosome/polylysine layers on the glass plate indicate that the supramolecular structures of self-aggregates of bacteriochlorophyll(BChl)- c in chlorosomes are maintained on the solid surface. BChl- c molecules could be completely extracted from a single chlorosome/polylysine layer on a glass plate as monomers in acetone. Visible absorption spectra of extracted pigments indicate that BChl- c molecules inside chlorosomes were not denatured by electrostatic adsorption of chlorosomes and that the amount of BChl- c molecules in chlorosomes adsorbed on a glass plate was (1.3 ± 0.05) × 10 − 10 mol/cm 2 (mean ± standard deviation, three measurements). The estimation of BChl- c amounts suggests that chlorosomes are not so densely immobilized on a polylysine-adsorbed quartz plate.

Bacteriochlorophyll-c Homolog Composition in Green Sulfur Photosynthetic Bacterium Chlorobium vibrioformeDependent on the Concentration of Sodium Sulfide in Liquid Cultures

Green sulfur photosynthetic bacteria Chlorobium (Chl.) vibrioforme (DSM 263 strain and NCIB 8327 substrain possessing BChl-c) and Chl. tepidum (ATCC 49652) were photoautotrophically grown in liquid cultures containing different concentrations of sodium sulfide (Na2S). BChl-c homologs possessing a methyl group at the 12-position tended to increase in cells of the two strains of Chl. vibrioforme cultured under high Na2S concentrations. In contrast, the Na2S concentration in liquid cultures did not affect the relative composition of BChl-c homologs in Chl. tepidum. 8-Propyl-12-methyl([P,M])-BChl-c homolog, which has been little observed in usual cultivations, could be isolated by reverse-phase high-performance liquid chromatography from the cells of Chl. vibrioforme grown under high Na2S contents. The [P,M]-BChl-c homolog has the R-configuration at the 3(1)-position, which was determined by 1H-NMR analyses.

Chlorosome Proteins Studied by MALDI-TOF-MS: Topology of CsmA in Chlorobium tepidum

Chlorosomes, the light-harvesting apparatus of green bacteria, are a unique antenna system, in which pigments are organized in aggregates rather than associated with proteins. Isolated chlorosomes from the green sulphur bacterium Chlorobium tepidum contain 10 surface-exposed proteins. Treatment of chlorosomes from Chlorobium tepidum with protease caused changes in the spectral properties of bacteriochlorophyll c and digestion of chlorosome proteins. Using SDS-PAGE analysis, immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) we have investigated the topology of the 59-residue CsmA protein. Our results show that at the N-terminus, the only amino acid available for protease degradation is the methionine. At the C-terminus, amino acids can be removed by protease treatment to produce a residual protein containing at least the sequence between residues 2 and 38. These results indicate that the N-terminal portion of the CsmA protein, which is predicted to be mainly hydrophobic, is buried in the chlorosome envelope.

Different Sensitivities to Oxygen Between Two Strains of the Photosynthetic Green Sulfur Bacterium Chlorobium vibrioforme NCIB 8327 with Bacteriochlorophyll c and d

Two sub-strains of the anoxygenic photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 were derived from the same clone and could be discriminated only by their possession of either bacteriochlorophyll (BChl) c or d as the major pigment in the peripheral light-harvesting antenna system, chlorosome (Saga Y et al. (2003) Anal Sci 19: 1575-1579). In the presence of a proper amount of oxygen in the initial culture medium, the BChl d strain showed longer retardation on its growth initiation than the BChl c strain, indicating that the latter was advantageous for survival under aerobic light conditions which produced reactive oxygen species in vivo. The result would be ascribable to the difference of the midpoint potentials between two kinds of chlorosomes formed by self-aggregates of BChl c and d as measured by their fluorescence quenching.

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium tepidum

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 +/- 17 nm x 66 +/- 13 nm x 4.3 +/- 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA-BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.

Seasonal Changes in the Structure of the Anoxygenic Phototrophic Bacterial Community in Lake Mogilnoe, a Relict Lake on Kil'din Island in the Barents Sea

An anaerobic phototrophic bacterial community in Lake Mogilnoe, a relict lake on Kil'din Island in the Barents Sea, was studied in June 1999 and September 2001. Irrespective of the season, the upper layer of the anaerobic zone of this lake had a specific species composition of sulfur phototrophic bacteria, which were dominated by the brown-colored green sulfur bacterium Chlorobium phaeovibrioides. The maximum number of phototrophic sulfur bacteria was observed in June 1999 at a depth of 9 m, which corresponded to a concentration of bacteriochlorophyll (Bchl) e equal to 4.6 mg/l. In September 2001, the maximum concentration of this pigment (3.4 mg/l) was found at a depth of 10 m. In both seasons, the concentration of Bchl a did not exceed 3 microg/l. Purple sulfur bacteria were low in number, which can be explained by their poor adaptation to the hydrochemical and optical conditions of the Lake Mogilnoe water. In June 1999, the water contained a considerable number of Pelodictyon phaeum microcolonies and Prosthecochloris phaeoasteroides cell chains, which was not the case in September 2001. A 16S rDNA-based phylogenetic analysis of pure cultures of phototrophic bacteria isolated from the lake water confirmed that the bacterial community is dominated by Chl. phaeovibrioides and showed the presence of three minor species, Thiocvstis gelatinosa, Thiocapsa sp., and Thiorhodococcus sp., the last of which is specific to Lake Mogilnoe.

Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit.

delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

Biogeochemical processes in the saline meromictic Lake Kaiike, Japan: implications from molecular isotopic evidences of photosynthetic pigments

Stable carbon and nitrogen isotopic compositions were determined for individual photosynthetic pigments isolated and purified from the saline meromictic Lake Kaiike, Japan, to investigate species-independent biogeochemical processes of photoautotrophs in the natural environment. In the anoxic monimolimnion and benthic microbial mats, the carbon isotopic compositions of BChls e and isorenieratene related to brown-coloured strains of green sulfur bacteria are substantially ( approximately 10 per thousand) depleted in (13)C relative to those found in the chemocline. In conjunction with 16S rDNA evidence reported previously, it strongly suggests that Pelodyctyon luteolum inhabited and photosynthesized in the anoxic monimolimnion and benthic microbial mats by using (13)C-depleted regenerated CO(2). By contrast, both Chl a and BChl a in the monimolimnion and microbial mats have similar isotopic compositions as they do in the chemocline, implying that the source organisms live only in the chemocline. In the chemocline, the nitrogen isotopic compositions of BChl e homologues ranges from -7.7 to-6.5 per thousand, whereas that of BChl a is -2.1 per thousand. These isotopic compositions suggest that green sulfur bacteria Chlorobium phaeovibrioides would conduct nitrogen fixation in the chemocline, whereas purple sulfur bacteria Halochromatium sp. and cyanobacteria Synechococcus sp. may assimilate nitrite.

Function of a PscD Subunit in a Homodimeric Reaction Center Complex of the Photosynthetic Green Sulfur Bacterium Chlorobium tepidum Studied by Insertional Gene Inactivation: REGULATION OF ENERGY TRANSFER AND FERREDOXIN-MEDIATED NADP+ REDUCTION ON THE CYTOPLASMIC SIDE

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.

Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum.

The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.

Lamellar Organization of Pigments in Chlorosomes, the Light Harvesting Complexes of Green Photosynthetic Bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations ∼20 Å apart. X-ray scattering from chlorosomes exhibited a feature with the same ∼20 Å spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.

Comparison between chlorosomes containing bacteriochlorophyll- c and chlorosomes containing bacteriochlorophyll- d isolated from two substrains of green sulfur photosynthetic bacterium Chlorobium vibrioforme NCIB 8327

Chlorosomes containing bacteriochlorophyll(BChl)- c and those containing BChl- d were isolated from two substrains of Chlorobium vibrioforme f. sp. thiosulfatophilum NCIB 8327, respectively. The two types of chlorosomes were investigated from the following aspect, what kinds of effects the molecular structure of chlorosomal BChls had on structural and spectroscopic properties of in vivo self-aggregates in chlorosomes without alteration of the other components such as chlorosomal proteins and lipids; both chlorosomes were expected to have the same components except for light-harvesting BChls. In their visible absorption spectra, the differences of Soret and Q y peak positions between BChl- c containing and BChl- d containing chlorosomes were similar to the differences between monomeric BChl- c and d. An inverse S-shaped CD signal in the Q y region of BChl- d containing chlorosomes was 1.4 times larger than that of BChl- c containing chlorosomes, when the Q y absorbance of the two chlorosomes was almost the same. This implies that the excitonic interaction of BChl- d is larger than that of BChl- c in natural chlorosomes. Resonance Raman spectroscopy showed that BChl self-assemblies in both chlorosomes were essentially formed by the same local structural interaction among 3 1-hydroxy group, 13-keto group, and central magnesium. BChl- d self-aggregates in chlorosomes were more tolerant of 1-hexanol than in vivo BChl- c aggregates, suggesting that the molecular structure of BChl- d provided more stable self-assemblies than BChl- c in natural chlorosomes.

Clustering of Genes with Function in the Biosynthesis of Bacteriochlorophyll and Heme in the Green Sulfur Bacterium Chlorobium Vibrioforme

identical to the pathway of heme biosynthesis (Fig. 1). The formation of 5- aminolevulinate can be achieved either from glycine and succinyl-CoA, in a reaction catalyzed by the enzyme 5-aminolevulinic acid synthase, or by a three step pathway from glutamate catalyzed by three enzymes, of which the second is glutamyl- tRNA dehydrogenase ( BEALE 1995). The succinyl- CoA pathway is used by nonplant eukaryotes and a group of bacteria that includes the purple photo- synthetic bacteria

Structure and organization of a 25 kbp region of the genome of the photosynthetic green sulfur bacterium Chlorobium vibrioforme containing Mg-chelatase encoding genes.

A region comprising approximately 25 kbp of the genome of the strictly anaerobic and obligate photosynthetic green sulfur bacterium Chlorobium vibrioforme has been mapped, subcloned and partly sequenced. Approximately 15 kbp have been sequenced in it's entirety and three genes with significant homology and feature similarity to the bchI, -D and -H genes and the chlI, -D and -H genes of Rhodobacter and Synechocystis strain PCC6803, respectively, which encode magnesium chelatase subunits, have been identified. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX, and is the first enzyme unique to the (bacterio)chlorophyll specific branch of the porphyrin biosynthetic pathway. The organization of the three Mg-chelatase encoding genes is unique to Chlorobium and suggests that the magnesium chelatase of C. vibrioforme is encoded by a single operon. The analyzed 25 kbp region contains five additional open reading frames, two of which display significant homology and feature similarity to genes encoding lipoamide dehydrogenase and genes with function in purine synthesis, and another three display significant homology to open reading frames with unknown function in distantly related bacteria. Putative E. coli sigma 70-like promoter sequences, ribosome binding sequences and rho-independent transcriptional stop signals within the sequenced 15 kbp region are related to the identified genes and orfs. Southern analysis, restriction mapping and partial sequencing of the remaining ca. 10 kbp of the analyzed 25 kbp region have shown that this part includes the hemA, -C, -D and -B genes (MOBERG and AVISSAR 1994), which encode enzymes with function in the early part of the biosynthetic pathway of porphyrins.

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes.

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.

Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation.

Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism. This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum by natural transformation with synthetic DNA constructs. Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented. These methods are ligation of DNA fragments with T4 DNA ligase, and megaprimer PCR.