Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light-dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.     

Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus

The hair‐like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo‐proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm‐promoting function in type IV pili‐producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non‐piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well‐studied type IV pili‐producing heterotrophic bacteria.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria

Direct conversion of carbon dioxide into chemicals using engineered autotrophic microorganisms offers a potential solution for both sustainability and carbon mitigation. Butyrate is an important chemical used in various industries, including fragrance, food, and plastics. A model cyanobacterium Synechococcus elongatus PCC 7942 was engineered for the direct photosynthetic conversion of CO ₂ to butyrate. An engineered Clostridium Coenzyme A (CoA)-dependent pathway leading to the synthesis of butyryl-CoA, the precursor to butyrate, was introduced into S. elongatus PCC 7942. Two CoA removal strategies were then individually coupled to the modified CoA-dependent pathway to yield butyrate production. Similar results were observed between the two CoA removal strategies. The best butyrate producing strain of S. elongatus resulted in an observed butyrate titer of 750 mg/L and a cumulative titer of 1.1 g/L. These results demonstrated the feasibility of photosynthetic butyrate production and expanded the chemical repertoire accessible for production by photoautotrophs.     

Diel Infection of a Cyanobacterium by a Contractile Bacteriophage

Light was found to strongly influence the infection of a freshwater cyanobacterium (Synechococcus elongatus PCC 7942) by a contractile DNA phage named AS-1. Phage progeny production was correlated with the amount of light in the laboratory and occurred in a diel pattern under natural light. At least one effect of light on AS-1 infection is at the level of adsorption.     

The ChrA homologue from a sulfur-regulated gene cluster in cyanobacterial plasmid pANL confers chromate resistance

The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell’s cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.     

Transformation of the fresh water cyanobacterium Synechococcus PCC7942 with the shuttle-vector pAQ-EX1 developed for the marine cyanobacterium Synechococcus PCC7002

The transformation of the fresh water cyanobacterium Synechococcus PCC7942 with the shuttle-vector pAQ-EX1 developed for the marine cyanobacterium S. PCC7002 was examined. The S. PCC7942 cells were successfully transformed with the pAQ-EX1 vector, and the vector was stably maintained in the transformant cells.     

Cell surface composition,released polysaccharides,and ionic strength mediate fast sedimentation in the cyanobacterium Synechococcus elongatus PCC 7942

Cyanobacteria are photosynthetic prokaryotes of high ecological and biotechnological relevance that have been cultivated in laboratories around the world for more than 70 years. Prolonged laboratory culturing has led to multiple microevolutionary events and the appearance of a large number of ‘domesticated’ substrains among model cyanobacteria. Despite its widespread occurrence, strain domestication is still largely ignored. In this work we describe Synechococcus elongatus PCC 7942-KU, a novel domesticated substrain of the model cyanobacterium S. elongatus PCC 7942, which presents a fast-sedimenting phenotype. Under higher ionic strengths the sedimentation rate increased leading to complete sedimentation in just 12 h. Through whole genome sequencing and gene deletion, we demonstrated that the Group 3 alternative sigma factor F plays a key role in cell sedimentation. Further analysis showed that significant changes in cell surface structures and a three-fold increase in released polysaccharides lead to the appearance of a fast-sedimenting phenotype. This work sheds light on the determinants of the planktonic to benthic transitions and provides genetic targets to generate fast-sedimenting strains that could unlock cost-effective cyanobacterial harvesting at scale.     

Role of the Escherichia coli glgX gene in glycogen metabolism

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.     

Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation

A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypicalty to complement a phoRcreC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29012Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate-limitation in the medium. These results imply that the gene products identified in this study are probably involved, either directly or indirectly, in the signal-transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (S ynechococcusph osphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.     

Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPF_T-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPF_F nor MPF_I could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPF_T-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes.     

Nucleotide sequence of the narB gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea

The nucleotide sequence of the structural gene of nitrate reductase (narB) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The narB gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases.     

Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs'' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.     

Sequence Determinants of Circadian Gene Expression Phase in Cyanobacteria

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits global biphasic circadian oscillations in gene expression under constant-light conditions. Class I genes are maximally expressed in the subjective dusk, whereas class II genes are maximally expressed in the subjective dawn. Here, we identify sequence features that encode the phase of circadian gene expression. We find that, for multiple genes, an ∼70-nucleotide promoter fragment is sufficient to specify class I or II phase. We demonstrate that the gene expression phase can be changed by random mutagenesis and that a single-nucleotide substitution is sufficient to change the phase. Our study provides insight into how the gene expression phase is encoded in the cyanobacterial genome.     

Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria

Photosynthetically derived fuels, such as those produced by microalgae, are touted as a future renewable energy source and a means for achieving energy independence. Realization of these claims, however, will require fuel production rates beyond the native capabilities of these microorganisms. The development of a metabolic engineering toolkit for microalgae will be key for reaching the production rates necessary for fuel production. This work advances the toolkit for cyanobacterial fuels by exploring the use of eukaryotic algal gene sources for free fatty acid biosynthesis rather than the traditional bacterial and plant sources. Many species of eukaryotic algae naturally accumulate high levels of triacylglycerol, a compound requiring three fatty acid side chains. Triacylglycerol accumulation implies that eukaryotic algae have naturally efficient enzymes for free fatty acid production, representing an unexplored resource for metabolic engineering targets. In this work, the model cyanobacterium, Synechococcus elongatus PCC7942, was engineered for free fatty acid production by targeting three main rate-limiting steps: (1) fatty acid release, catalyzed by a thioesterase, (2) fixation of carbon by ribulose-1,5-bisphosphate carboxylase/oxygenase, and (3) the first committed step in fatty acid biosynthesis, acetyl-CoA carboxylase. The recombinant acyl-ACP thioesterase and acetyl-CoA carboxylase were derived from the model green alga, Chlamydomonas reinhardtii CC-503. By targeting these proposed rate-determining steps, free fatty acid production was improved on a cell weight basis; however, the overall concentration of excreted free fatty acid did not increase. Recombinant gene expression was optimized by using native promoters, and while expression improved, the free fatty acid yield did not likewise increase. From physiological measurements, it was determined that free fatty acid production in S. elongatus PCC7942 is ultimately limited by the negative physiological effects associated with free fatty acid synthesis rather than bottlenecks within the metabolic pathway. This work demonstrates the successful expression of algal genes in a cyanobacterial host, but further improvement in free fatty acid yields will only be possible when the negative effects of free fatty acid production are mitigated.     

Role of cyanobacterial phosphoketolase in energy regulation and glucose secretion under dark anaerobic and osmotic stress conditions

Primary metabolism in cyanobacteria is built on the Calvin-Benson-Bassham (CBB) cycle, oxidative pentose phosphate (OPP) pathway, Embden–Meyerhof–Parnas (EMP) pathway, and the tricarboxylic acid (TCA) cycle. Phosphoketolase (Xpk), commonly found in cyanobacteria, is an enzyme that is linked to all these pathways. However, little is known about its physiological role. Here, we show that most of the cyanobacterial Xpk surveyed are inhibited by ATP, and both copies of Xpk in nitrogen-fixing Cyanothece ATCC51142 are further activated by ADP, suggesting their role in energy regulation. Moreover, Xpk in Synechococcus elongatus PCC7942 and Cyanothece ATCC51142 show that their expressions are dusk-peaked, suggesting their roles in dark conditions. Finally, we find that Xpk in S. elongatus PCC7942 is responsible for survival using ATP produced from the glycogen-to-acetate pathway under dark, anaerobic condition. Interestingly, under this condition, xpk deletion causes glucose secretion in response to osmotic shock such as NaHCO₃, KHCO₃ and NaCl as part of incomplete glycogen degradation. These findings unveiled the role of this widespread enzyme and open the possibility for enhanced glucose secretion from cyanobacteria.     

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion. Received: 28 July 1997 / Accepted: 26 November 1997