Nicholas F. Tsinoremas

A sigma factor that modifies the circadian expression of a subset of genes in cyanobacteria.

We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70‐like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942

In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp. strain PCC 7942

Extensive circadian (daily) control over gene expression in the cyanobacterium Synechococcus sp. strain PCC 7942 is programmed into at least two differentially phased groups. The transcriptional activity of the smaller group of genes is maximal at about dawn and minimal at about dusk. We identified one of the genes belonging to this latter group as purF, which encodes the key regulatory enzyme in the de novo purine synthetic pathway, glutamine PRPP amidotransferase (also known as amidophosphoribosyltransferase). Its expression pattern as a function of circadian time was confirmed by both luminescence from a purF:: luxAB reporter strain and the abundance of purF mRNA. By fusing sequences upstream of the purF coding region to promoterless luxAB genes, we identified a limited upstream region, which potentially regulates purF circadian expression patterns in vivo. We also identified the purL gene immediately upstream of purF. The purL gene encodes FGAM synthetase, the fourth enzyme in the purine nucleotide biosynthesis pathway. Although these genes are expressed as part of a larger operon in other bacteria, reporter gene fusions revealed that purF and purL are transcribed independently in Synechococcus and that they are expressed at different phases of the circadian cycle. This differential expression pattern may be related to the oxygen sensitivity of amidophosphoribosyltransferase.

General effect of photosynthetic electron transport inhibitors on translation precludes their use for investigating regulation of D1 biosynthesis in Synechococcus sp. strain PCC 7942

Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an explanation for conflicting reports regarding the role of photosynthetic electron transport in regulation of psbA expression and D1 interchange. Inhibitors that block at different points in the photosynthetic electron transport chain were administered and the effect on expression of psbAII, which encodes the high-light-induced form II of D1, was examined by measuring changes in transcript levels and in the activities of reporter enzymes. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PSII, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), an inhibitor of the cytochrome b6/f complex, prevented high-light-induced increase in β-galactosidase activity from a psbAII::lacZ gene fusion when added at a concentration that completely inhibits photosynthetic electron transport (1 μM). The same effect was observed for luciferase activity from transcriptional and translational fusions of psbAII to the luxAB genes from Vibrio harveyi. DCMU (1 μM) arrested luciferase expression at low-light levels – thus eliminating the high light response – whereas a sublethal concentration (50 nM), which reduces electron transport by 50%, had intermediate effects on psbAII-driven luciferase activity. However, psbAII transcript levels, monitored by northern blot analysis, were not altered by electron transport inhibitors, either at low-light intensity or following a high-light exposure. The suppressive effect of DCMU on expression of reporter enzymes was not restricted to the high-light response of psbAII-driven reporter systems, but was also observed using an isopropyl-(-d)-thiogalactopyranoside (IPTG)-inducible trc promoter fused to luxAB. This construct only marginally responded to IPTG addition when DCMU was present. Thus, blocking electron transport in Synechococcus affects the translation machinery in a general way, and the use of electron transport inhibitors is of limited value when focusing on specific redox regulation of D1 protein synthesis or degradation.

Expression of a higher-plant chloroplast psbD promoter in a cyanobacterium (Synechococcus sp. strain PCC7942) reveals a conserved cis-element, designated PGT, that differentially interacts with sequence-specific binding factors during leaf development

Abstract The chloroplast psbD gene, which encodes the D2 subunit of photosystem II, is regulated by a blue light-responsive promoter (BLRP). We tested the ability of different regions of the barley (Hordeum vulagare) BLRP to drive transcription of the lacZ reporter gene in genomic transformants of Synechococcus sp. strain PCC7942. The barley BLRP was transcribed in Synechococcus from the same initiation sites that are used in plant chloroplasts in vivo. A region of the BLRP, residing between –83 and –112 bp upstream from the transcription initiation sites, functioned as a negative element in Synechococcus. Nucleotide sequences within this region are conserved among the psbD genes of several monocots and dicots, and with the nuclear negative regulatory element GT. Thus this new cis-element was designated Plastid GT, PGT. Proteins from chloroplasts of barley and Arabidopsis thaliana interacted with PGT in a sequence-specific and developmental-dependent manner. The DNA-protein complexes from Arabidopsis chloroplasts are composed of 60- and 38-kDa polypeptides. We postulate that GT and PGT have evolved in the nucleus and chloroplast, respectively, from a common ancestral regulatory element.

Circadian Rhythms of Gene Expression in Cyanobacteria

The cyanobacteria are the simplest organisms known to exhibit circadian rhythms of various metabolic functions as a manifestation of an endogenous biological clock [1], The unicellular cyanobacterium Synechococcus sp. strain PCC 7942 provides an excellent model system with which to analyze the function of the circadian clock and its control over gene expression. Synechococcusis readily transformable, and sophisticated tools have been developed for its genetic manipulation [2-4]. Clock control of gene expression is easily demonstrated by using the Vibrio harveyi luxABgenes in transcriptional fusions with the promoters of Synechococcusgenes of interest [5]. The promoter of the psbAIgene, one of three genes that encodes the Dl protein of photosystem II, was fused to luxABand this reporter was recombined into the Synechococcusgenome. The resulting transformants produced light when provided with the long-chain aldehyde substrate of the enzyme (decanal) [5]. Entrainment of the cells to a 12 h light: 12 h darkness cycle (LD12:12), followed by incubation in continuous light, resulted in bioluminescence expressed rhythmically, peaking and troughing once per day, with a period of 24 h. The phase of the rhythm could be reset by pulses of darkness, and the period stayed very near to 24 h over a 10°C range of continuous growth temperatures. These features demonstrated that the rhythm of bioluminescence, as a function of psbAIexpression in Synechococcus,conforms to the criteria that have been used to define control by an endogenous circadian oscillator [5].