History of the ADP/ATP-translocase-encoding gene,a parasitism gene transferred from a Chlamydiales ancestor to plants 1 billion years ago

Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene, tlc, is found only in Rickettsiales, Chlamydiales, and plant and alga plastids. We demonstrate the presence of tlc in Parachlamydia acanthamoebae. This gene shares more similarity with the tlc1 gene of Chlamydiaceae and the tlc of plant and alga plastids than with the tlc2 gene of Chlamydiaceae. Phylogenetic analysis, including all other tlc homologs found in GenBank, showed that tlc was duplicated in a Chlamydiales ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by tlc. Plant and alga plastids acquired tlc approximately when Parachlamydiaceae and Chlamydiaceae diverged, at the eucaryotic radiation time, ca. 1 billion years ago.     

Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.     

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

Background

Chlamydiae species are of much importance from a clinical viewpoint. Their diversity both in terms of their numbers as well as clinical involvement are presently believed to be significantly underestimated. The obligate intracellular nature of chlamydiae has also limited their genetic and biochemical studies. Thus, it is of importance to develop additional means for their identification and characterization.

Results

We have carried out analyses of available chlamydiae genomes to identify sets of unique proteins that are either specific for all Chlamydiales genomes, or different Chlamydiaceae family members, or members of the Chlamydia and Chlamydophila genera, or those unique to Protochlamydia amoebophila, but which are not found in any other bacteria. In total, 59 Chlamydiales-specific proteins, 79 Chlamydiaceae-specific proteins, 20 proteins each that are specific for both Chlamydia and Chlamydophila and 445 ORFs that are Protochlamydia-specific were identified. Additionally, 33 cases of possible gene loss or lateral gene transfer were also detected.

Conclusion

The identified chlamydiae-lineage specific proteins, many of which are highly conserved, provide novel biomarkers that should prove of much value in the diagnosis of these bacteria and in exploration of their prevalence and diversity. These conserved protein sequences (CPSs) also provide novel therapeutic targets for drugs that are specific for these bacteria. Lastly, functional studies on these chlamydiae or chlamydiae subgroup-specific proteins should lead to important insights into lineage-specific adaptations with regards to development, infectivity and pathogenicity.     

Identification and Characterization of the Cassava Core-Clock Gene EARLY FLOWERING 4

The angiosperm circadian clock has been well established from molecular-genetic studies in a temperate plant model. Conservation of clock function is less explored in plants from the tropics. Cassava (Manihot esculenta) is a staple crop grown in the tropics that has been of limited research interest, and more generally, research on photoperiod and clock genes has been sparse. EARLY FLOWERING 4 (AtELF4) of the temperate plant Arabidopsis thaliana (Arabidopsis) has been reported to be required for photoperiod perception and circadian function. Here, we describe our start to identify circadian and photoperiod genes in cassava with an account on the characterization of its ELF4 gene (MeELF4). After isolating MeELF4, a phylogenetic study was conducted and it was found to cluster within the ELF4 subclade of the ELF4/EFL super-family. Similar to studies in temperate plants, MeELF4 was shown to be an evening-expressed gene in cassava. This collectively suggested to us that MeELF4 could be a functional ortholog of AtELF4. To test this, complementation studies of MeELF4 were performed in the Arabidopsis elf4 mutant. Hypocotyl-length measurements and flowering-time analysis were performed. MeELF4-complementation transgenics in the elf4 background were restored to the wild-type growth habit, suggesting a total rescue of photoperiodic perception. To expand on the molecular role of MeELF4 in the resulting transgenic-complementation lines, the CCA1 and CCR2 promoter-luciferase markers where respectively introduced and bioluminescence-imaging experiments revealed a restoration of circadian-regulated gene expression. The collective results showed that the cassava gene MeELF4 is a functional clock ortholog of AtELF4.     

Diversification of the Light-Harvesting Complex Gene Family via Intra- and Intergenic Duplications in the Coral Symbiotic Alga Symbiodinium

The light-harvesting complex (LHC) is an essential component in light energy capture and transduction to facilitate downstream photosynthetic reactions in plant and algal chloroplasts. The unicellular dinoflagellate alga Symbiodinium is an endosymbiont of cnidarian animals, including corals and sea anemones, and provides carbohydrates generated through photosynthesis to host animals. Although Symbiodinium possesses a unique LHC gene family, called chlorophyll a-chlorophyll c₂-peridinin protein complex (acpPC), its genome-level diversity and evolutionary trajectories have not been investigated. Here, we describe a phylogenetic analysis revealing that many of the LHCs are encoded by highly duplicated genes with multi-subunit polyprotein structures in the nuclear genome of Symbiodinium minutum. This analysis provides an extended list of the LHC gene family in a single organism, including 80 loci encoding polyproteins composed of 145 LHC subunits recovered in the phylogenetic tree. In S. minutum, 5 phylogenetic groups of the Lhcf-type gene family, which is exclusively conserved in algae harboring secondary plastids of red algal origin, were identified. Moreover, 5 groups of the Lhcr-type gene family, of which members are known to be associated with PSI in red algal plastids and secondary plastids of red algal origin, were identified. Notably, members classified within a phylogenetic group of the Lhcf-type (group F1) are highly duplicated, which may explain the presence of an unusually large number of LHC genes in this species. Some gene units were homologous to other units within single loci of the polyprotein genes, whereas intergenic homologies between separate loci were conspicuous in other cases, implying that gene unit ‘shuffling’ by gene conversion and/or genome rearrangement might have been a driving force for diversification. These results suggest that vigorous intra- and intergenic gene duplication events have resulted in the genomic framework of photosynthesis in coral symbiont dinoflagellate algae.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.     

A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera

Corals exhibit circadian behaviors, but little is known about the molecular mechanisms underlying the regulation of these behaviors. We surveyed the recently decoded genome of the coral, Acropora digitifera, for photoreceptor and circadian genes, using molecular phylogenetic analyses. Our search for photoreceptor genes yielded seven opsin and three cryptochrome genes. Two genes from each family likely underwent tandem duplication in the coral lineage. We also found the following A. digitifera orthologs to Drosophila and mammalian circadian clock genes: four clock, one bmal/cycle, three pdp1-like, one creb/atf, one sgg/zw3, two ck2alpha, one dco (csnk1d/cnsk1e), one slim/BTRC, and one grinl. No vrille, rev-ervα/nr1d1, bhlh2, vpac2, adcyap1, or adcyaplr1 orthologs were found. Intriguingly, in spite of an extensive survey, we also failed to find homologs of period and timeless, although we did find one timeout gene. In addition, the coral genes were compared to orthologous genes in the sea anemone, Nematostella vectensis. Thus, the coral and sea anemone genomes share a similar repertoire of circadian clock genes, although A. digitifera contains more clock genes and fewer photoreceptor genes than N. vectensis. This suggests that the circadian clock system was established in a common ancestor of corals and sea anemones, and was diversified by tandem gene duplications and the loss of paralogous genes in each lineage. It will be interesting to determine how the coral circadian clock functions without period.     

Palmitate Inhibits SIRT1-Dependent BMAL1/CLOCK Interaction and Disrupts Circadian Gene Oscillations in Hepatocytes

Elevated levels of serum saturated fatty acid palmitate have been shown to promote insulin resistance, increase cellular ROS production, and trigger cell apoptosis in hepatocytes during the development of obesity. However, it remains unclear whether palmitate directly impacts the circadian clock in hepatocytes, which coordinates nutritional inputs and hormonal signaling with downstream metabolic outputs. Here we presented evidence that the molecular clock is a novel target of palmitate in hepatocytes. Palmitate exposure at low dose inhibits the molecular clock activity and suppresses the cyclic expression of circadian targets including Dbp, Nr1d1 and Per2 in hepatocytes. Palmitate treatment does not seem to alter localization or reduce protein expression of BMAL1 and CLOCK, the two core components of the molecular clock in hepatocytes. Instead, palmitate destabilizes the protein-protein interaction between BMAL1-CLOCK in a dose and time-dependent manner. Furthermore, we showed that SIRT1 activators could reverse the inhibitory action of palmitate on BMAL1-CLOCK interaction and the clock gene expression, whereas inhibitors of NAD synthesis mimic the palmitate effects on the clock function. In summary, our findings demonstrated that palmitate inhibits the clock function by suppressing SIRT1 function in hepatocytes.     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.     

Functional Characterization of the Chlamydomonas reinhardtii ERG3 Ortholog,a Gene Involved in the Biosynthesis of Ergosterol

Background

The predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Higher plants and fungi synthesize sterols by different pathways, with plants producing cycloartenol as a precursor to end-product sterols, while non-photosynthesizing organisms like yeast and humans produce lanosterol as a precursor. Analysis of the C. reinhardtii genome sequence reveals that this algae is also likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome.

Methodology/Principal Findings

A gene with homology to the yeast sterol C-5 desaturase, ERG3, is present in the Chlamydomonas genome. To test whether the ERG3 ortholog of C. reinhardtii encodes a sterol C-5 desaturase, Saccharomyces cerevisiae ERG3 knockout strains were created and complemented with a plasmid expressing the Chlamydomonas ERG3. Expression of C. reinhardtii ERG3 cDNA in erg3 null yeast was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function.

Conclusions/Significance

Complementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase.