Proteins In Synechocystis Sp Research Articles

Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

Quantifying cellular components is a basic and important step for understanding how a cell works, how it responds to environmental changes, and for re-engineering cells to produce valuable metabolites and increased biomass. We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. Components of biosynthetic pathways, such as those for chlorophyll or for photosystem II assembly, range between 1000 and 10,000 copies per cell, but can be tenfold higher for CO2 fixation enzymes. The most abundant subunits are those for photosystem I, with around 100,000 copies per cell, approximately 2 to fivefold higher than for photosystem II and ATP synthase, and 5–20 fold more than for the cytochrome b6f complex. Disparities between numbers of pathway enzymes, between components of electron transfer chains, and between subunits within complexes indicate possible control points for biosynthetic processes, bioenergetic reactions and for the assembly of multisubunit complexes.

Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

BackgroundBased on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl–acyl carrier protein synthetase), and surface layer protein (encoded by sll1951).ResultsThree Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG11-half N and BG11-N conditions, with the exception of strain KS. Under BG11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG11-N growth medium. This strain KS significantly acclimated to the BG11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion.ConclusionsMutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Production of glycogen, PHB, biohydrogen, NAD(P)H, and proteins in Synechocystis sp. PCC 6803 disrupted in metabolically linked biosynthetic pathway(s)

Production of glycogen, PHB, biohydrogen, NAD(P)H, and proteins in Synechocystis sp. PCC 6803 disrupted in metabolically linked biosynthetic pathway(s)

The Role of Outer Membrane Protein(s) Harboring SLH/OprB-Domains in Extracellular Vesicles' Production in Synechocystis sp. PCC 6803.

Cyanobacteria are a group of photosynthetic prokaryotes that contribute to primary production on a global scale. These microorganisms release vesicles to the extracellular environment, spherical nanosized structures, derived essentially from the outer membrane. Even though earlier works in model Gram-negative bacteria have hypothesized that outer membrane stability is crucial in vesicle formation, the mechanisms determining vesicle biogenesis in cyanobacteria remain unknown. Here, we report on the identification of six candidate genes encoding outer membrane proteins harboring SLH/OprB-domains in the genome of the model cyanobacterium Synechocystis sp. PCC 6803. Using a genetics-based approach, one gene was found to encode an essential protein (Slr1841), while the remaining five are not essential for growth under standard conditions. Vesicle production was monitored, and it was found that a mutant in the gene encoding the second most abundant SLH/OprB protein in Synechocystis sp. PCC 6803 outer membrane (Slr1908) produces more vesicles than any of the other tested strains. Moreover, the Slr1908-protein was also found to be important for iron uptake. Altogether, our results suggest that proteins containing the SLH/OprB-domains may have dual biological role, related to micronutrient uptake and to outer membrane stability, which, together or alone, seem to be involved in cyanobacterial vesicle biogenesis.

Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO2, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (FAFB), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s−1). These data unambiguously show that reduced ferredoxin, or possibly reduced FAFB, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (FAFB) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.

Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp. PCC 6803

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.

Deciphering proteolysis pathways for the error-prone DNA polymerase in cyanobacteria.

Protein quality control pathways require AAA+ proteases, such as Clp and Lon. Lon protease maintains UmuD, an important component of the error-prone DNA repair polymerase (Pol V), at very low levels in E. coli. Most members of the phylum Cyanobacteria lack Lon (including the model cyanobacterium, Synechocystis sp. PCC6803), so maintenance of UmuD at low levels must employ different proteases. We demonstrate that the first 19 residues from the N-terminus of UmuD (Sug1-19 ) fused to a reporter protein are adequate to trigger complete proteolysis and that mutation of a single leucine residue (L6) to aspartic acid inhibits proteolysis. This process appears to follow the N-end rule and is mediated by ClpA/P protease and the ClpS adaptor. Additionally, mutations of arginine residues in the Sug1-19 tag suggest that the ClpX/P pathway also plays a role in proteolysis. We propose that there is a dual degron at the N-terminus of the UmuD protein in Synechocystis sp. PCC6803, which is distinct from the degron required for degradation of UmuD in E. coli. The use of two proteolysis pathways to tune levels of UmuD might reflect how a photosynthetic organism responds to multiple environmental stressors.

Proteome Mapping of a Cyanobacterium Reveals Distinct Compartment Organization and Cell-Dispersed Metabolism.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal membrane system that fulfills the energetic requirements of the cell.

A global network-based protocol for functional inference of hypothetical proteins in Synechocystis sp. PCC 6803

Functional inference of hypothetical proteins (HPs) is a significant task in the post-genomic era. We described here a network-based protocol for functional inference of HPs using experimental transcriptomic, proteomic, and protein–protein interaction (PPI) datasets. The protocol includes two steps: i) co-expression networks were constructed using large proteomic or transcriptomic datasets of Synechocystis sp. PCC 6803 under various stress conditions, and then combined with a Synechocystis PPI network to generate bi-colored networks that include both annotated proteins and HPs; ii) a global algorithm was adapted to the bi-colored networks for functional inference of HPs. The algorithm ranked the associations between genes/proteins with known GO functional categories, and assumed that the top one ranked HP for each GO functional category might have a function related to the GO functional category. We applied the protocol to all HPs of the model cyanobacterium Synechocystis, and were able to assign putative functions to 122 HPs that have never been functionally characterized previously. Finally, the functional inference was validated by the known biological information of operon, and results showed that more than 70% HPs could be correctly validated. The study provided a new protocol to integrate different types of OMICS datasets for functional inference of HPs, and could be useful in achieving new insights into the Synechocystis metabolism.

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter Ptrc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.

Systematic characterization of hypothetical proteins in Synechocystis sp. PCC 6803 reveals proteins functionally relevant to stress responses

We described here a global detection and functional inference of hypothetical proteins involved in stress response in Synechocystis sp. PCC 6803. In the study, we first applied an iTRAQ-LC–MS/MS based quantitative proteomics to the Synechocystis cells grown under five stress conditions. The analysis detected a total of 807 hypothetical proteins with high confidence. Among them, 480 were differentially regulated. We then applied a Weighted Gene Co-expression Network Analysis approach to construct transcriptional networks for Synechocystis under nutrient limitation and osmotic stress conditions using transcriptome datasets. The analysis showed that 305 and 467 coding genes of hypothetical proteins were functionally relevant to nutrient limitation and osmotic stress, respectively. A comparison of responsive hypothetical proteins to all stress conditions allowed identification of 22 hypothetical proteins commonly responsive to all stresses, suggesting they may be part of the core stress responses in Synechocystis. Finally, functional inference of these core stress responsive proteins using both sequence similarity and non-similarity approaches was conducted. The study provided new insights into the stress response networks in Synechocystis, and also demonstrated that a combination of experimental “OMICS” and bioinformatics methodologies could improve functional annotation for hypothetical proteins.

Specific and promiscuous functions of multiple DnaJ proteins in Synechocystis sp. PCC 6803

Cyanobacterial genomes typically encode multiple Hsp70 (DnaK) and Hsp40 (DnaJ) chaperones, and in the genome of the cyanobacterium Synechocystis PCC 6803, three DnaK proteins are encoded together with seven DnaJ proteins. While only two of the DnaJ proteins can complement the growth defect of an Escherichia coli ΔdnaJ strain, only disruption of the dnaJ gene sll0897 resulted in a growth defect at elevated temperatures. Based on the domain structure and the phenotype observed following disruption of the encoding gene, Sll0897 can be classified as a canonical heat-shock protein in Synechocystis. Furthermore, most dnaJ genes could be deleted individually, whereas disruption of the gene encoding the DnaJ Sll1933 failed, which suggests an essential, yet undefined, function for Sll1933. Since after deletion of the remaining dnaJ genes the phenotypes were not altered, the functions of these DnaJs either are not critical or are taken over by the remaining DnaJs. Nevertheless, only the two dnaJ genes sll0909 and sll1384 could be disrupted in combination, suggesting physiological functions for the two encoded proteins which either are not overlapping and/or can be fulfilled by the remaining DnaJs in the double-disruption strain. Taken together, the present analysis indicates specific and promiscuous functions for multiple DnaJ proteins in Synechocystis.

Engineered ectopic expression of the psbA gene encoding the photosystem II D1 protein in Synechocystis sp. PCC6803

A genetic vector-recipient system was developed to engineer expression of the wild-type psbA2 gene encoding the photosystem II (PSII) D1 protein only from a non-native location (ectopic) in the Synechocystis sp. PCC6803 and the result was a new strain, designated MK1. While MK1 accumulates near normal levels of PSII under low light conditions, it is very sensitive to photoinhibition. This phenotype can be traced to impaired PSII repair capacity. Based upon the hypothesis that the non-native transcriptional activity of the re-introduced psbA gene was insufficient to sustain the translation rate necessary for normal PSII repair rates, we conducted a quantitative analysis of the relationship between psbA transcript abundance on the rate of recovery from photoinhibition. Analysis of MK1 and two other engineered strains, with intermediate levels of psbA mRNA, indicated that transcript levels are indeed limiting the engineered strains. Furthermore, transcript levels may become limiting even in the wild-type, but only under very high light conditions when the demands for D1 replacement synthesis are maximal. The work extends the original studies of Komenda and colleagues (Komenda et al. (2000) Plant Mol Biol 42(4):635-645) and sets the stage for alternative approaches to engineering non-native expression of the D1 protein.

Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

The PII signaling protein plays a pivotal role in the coordination of carbon and nitrogen metabolism in a wide variety of bacteria, Archaea, and plant chloroplasts. By using a yeast two-hybrid screening system, we identified a transmembrane protein, designated PamA (encoded by sll0985), as a PII-binding protein in Synechocystis sp. PCC 6803. The interaction between PII and PamA was confirmed in vitro, and the interaction was inhibited in the presence of ATP and 2-oxoglutarate, whereas the interaction was not influenced by the phosphorylation status of PII. Northern blot analyses revealed that the transcripts of a set of nitrogen-related genes, including nblA, nrtABCD, and ureG, were decreased in a pamA deletion mutant. The mRNA and protein levels of a group 2 sigma factor SigE were also reduced by the pamA mutation, and transcripts for sugar catabolic genes, such as gap1, zwf, and gnd that are under the control of SigE, were consequently decreased in the pamA mutant. In addition, the pamA mutant was found to be unable to grow in glucose-containing media. These results indicate that PamA has a role in the transcript control of genes for nitrogen and sugar metabolism in Synechocystis sp. PCC 6803.

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl- - or Ca2+ -limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and DeltaPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the DeltaPsbO: DeltaPsbP, DeltaPsbQ:DeltaPsbP, DeltaPsbU:DeltaPsbP and DeltaPsbV:DeltaPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the DeltaPsbO:DeltaPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932-938]. However, thermostability and thermal acclimation in DeltaPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with approximately 3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164-2175].

The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen.

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.

The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20°C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44°C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.