Cyanobacteria have evolved a number of acclimation strategies to sense and respond to changing nutrient and light conditions. Leptolyngbya sp. JSC-1 was recently shown to photoacclimate to far-red light by extensively remodeling its photosystem (PS) I, PS II and phycobilisome complexes, thereby gaining the ability to grow in far-red light. A 21-gene photosynthetic gene cluster (rfpA/B/C, apcA2/B2/D2/E2/D3, psbA3/D3/C2/B2/H2/A4, psaA2/B2/L2/I2/F2/J2) that is specifically expressed in far-red light encodes the core subunits of the three major photosynthetic complexes. The growth responses to far-red light were studied here for five additional cyanobacterial strains, each of which has a gene cluster similar to that in Leptolyngbya sp. JSC-1. After acclimation all five strains could grow continuously in far-red light. Under these growth conditions each strain synthesizes chlorophylls d, f and a after photoacclimation, and each strain produces modified forms of PS I, PS II (and phycobiliproteins) that absorb light between 700 and 800 nm. We conclude that these photosynthetic gene clusters are diagnostic of the capacity to photoacclimate to and grow in far-red light. Given the diversity of terrestrial environments from which these cyanobacteria were isolated, it is likely that FaRLiP plays an important role in optimizing photosynthesis in terrestrial environments.

Chlorophyll f (Chl f) permits some cyanobacteria to expand the spectral range for photosynthesis by absorbing far-red light. We used reverse genetics and heterologous expression to identify the enzyme for Chl f synthesis. Null mutants of "super-rogue" psbA4 genes, divergent paralogs of psbA genes encoding the D1 core subunit of photosystem II, abolished Chl f synthesis in two cyanobacteria that grow in far-red light. Heterologous expression of the psbA4 gene, which we rename chlF, enables Chl f biosynthesis in Synechococcus sp. PCC 7002. Because the reaction requires light, Chl f synthase is probably a photo-oxidoreductase that employs catalytically useful Chl a molecules, tyrosine YZ, and plastoquinone (as does photosystem II) but lacks a Mn4Ca1O5 cluster. Introduction of Chl f biosynthesis into crop plants could expand their ability to use solar energy.

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.

Terrestrial cyanobacteria often occur in niches that are strongly enriched in far-red light (FRL; λ > 700 nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRL photoacclimation (FaRLiP). During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS) I, PS II, and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d. Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB, and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203. The resulting mutants were no longer able to modify their photosynthetic apparatus to absorb FRL, were no longer able to synthesize Chl f, inappropriately synthesized Chl d in white light, and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.

The Jacob Blaustein lnstitute for Desert Research, Ben-Gurion University of the Negev, Sede-Boker 84990, ISiaelTo design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 pE m+ sec-l), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 pE m-2 sec-l (doubling time -28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-IesslapcE-strain grew photoheterotrophically at normal light intensity (50 pE m-2 se&) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE-strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-IesslapcE-strains had an approximately sixfold enrichment of PSll on a chlorophyll basis and were as active in oxygen evolution (on a per PSll basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSll studies and as background strains to analyze site-and region-directed PSll mutants in vivo.

Phototrophic organisms are superbly adapted to different light environments but often must acclimate to challenging competition for visible light wavelengths in their niches. Some cyanobacteria overcome this challenge by expressing paralogous photosynthetic proteins and by synthesizing and incorporating ~8% chlorophyll f into their Photosystem I (PSI) complexes, enabling them to grow under far-red light (FRL). We solved the structure of FRL-acclimated PSI from the cyanobacterium Fischerella thermalis PCC 7521 by single-particle, cryo–electron microscopy to understand its structural and functional differences. Four binding sites occupied by chlorophyll f are proposed. Subtle structural changes enable FRL-adapted PSI to extend light utilization for oxygenic photosynthesis to nearly 800 nm. This structure provides a platform for understanding FRL-driven photosynthesis and illustrates the robustness of adaptive and acclimation mechanisms in nature.

The suf operon is composed of four genes (sufB, sufC, sufD, and sufS) and is highly conserved in the genomes of cyanobacteria. Open reading frame sll0088 in Synechocystis sp. strain PCC 6803 is located near the 5 end of the suf operon but is transcribed in the direction opposite that of the suf operon. We previously reported the isolation of two independent suppressor strains of C14S PsaC that mapped to sll0088 and restored photoautotrophic growth. The protein encoded by sll0088 has two significant features: (i) a DNA-binding domain near the N terminus and (ii) four highly conserved cysteine residues near the C terminus. The protein has high sequence similarity to transcription regulatory proteins with a conserved DNA-binding domain and can be classified in the DeoR family of helix-loop-helix proteins. The protein falls into a further subclass that contains a C-X 12 -C-X 13 -C-X 14 -C motif near the C terminus, which may represent a metal-binding site. The expressed Sll0088 protein harbored an iron-sulfur cluster as shown by optical and electron paramagnetic resonance spectroscopy. Compared to the wild type, expression levels of the sufBCDS genes were elevated when cells were grown under conditions of oxidative and iron stress and were even higher in a null mutant of Synechococcus sp. strain PCC 7002 in which the sll0088 homolog was insertionally inactivated. In agreement with the proposed role of the sufBCDS genes in iron metabolism, the growth rate of the null mutant was significantly higher than that of the wild type under iron-limiting conditions. We propose that the protein encoded by sll0088 is a transcriptional repressor of the suf operon, and we name the gene sufR.The biogenesis and assembly of fully functional photosystem I (PS I) require the assembly of F X , F A , and F B , which are [4Fe-4S] clusters, and soluble ferredoxin, which contains a [2Fe-2S] cluster. Iron-sulfur clusters can be assembled and inserted into proteins in vitro by incubating the apoprotein with iron, sulfide, and a thiol-containing reducing agent, such as 2-mercaptoethanol (22). This approach has been used in conjunction with bacterial expression systems to elucidate the structure and function of a variety of photosynthetic iron-sulfur proteins, including PsaC (reviewed in reference 11). In contrast, the in vivo biosynthesis of iron-sulfur clusters in photosynthetic complexes involves biochemical assembly processes that are poorly characterized. One known factor is a membrane-bound rubredoxin, which appears to be associated exclusively with the assembly of the F X iron-sulfur cluster (33, 34). Another known factor is the open reading frame sll0088 in Synechocystis sp. strain PCC 6803, which appears to have a role in regulating the biogenesis of PS I (43) and is the topic of this study.In nonphotosynthetic organisms, iron-sulfur cluster assembly is known to be a multistep process that involves cluster biosynthesis, insertion, and stabilization (9). The isc operon is implicated in generalized iron-sulfur cluster assembly in many or...