Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope.

The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone's interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.

Dual stoichiometry and subunit organization in the ClpP1/P2 protease from the cyanobacterium Synechococcus elongatus.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. To investigate the proteolytic core of the ClpXP1/P2 protease from the cyanobacterium Synechococcus elongatus we have used a non-denaturing mass spectrometry approach. We show that the proteolytic core is a double ring tetradecamer consisting of an equal number of ClpP1 and ClpP2 subunits with masses of 21.70 and 23.44 kDa, respectively. Two stoichiometries are revealed for the heptameric rings: 4ClpP1+3ClpP2 and 3ClpP1+4ClpP2. When combined in the double ring the stoichiometries are (4ClpP1+3ClpP2)+(3ClpP1+4ClpP2) and 2×(3ClpP1+4ClpP2) with a low population of a 2×(4ClpP1+3ClpP2) tetradecamer. The assignment of the stoichiometries is confirmed by collision-induced dissociation of selected charge states of the intact heptamer and tetradecamer. Presence of the heterodimers, heterotetramers and heterohexamers, and absence of the mono-oligomers, in the mass spectra of the partially denatured protease indicates that the ring complex consists of a chain of ClpP1/ClpP2 heterodimers with the ring completed by an additional ClpP1 or ClpP2 subunit.

Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

Assembly of the chloroplast ATP-dependent Clp protease in Arabidopsis is regulated by the ClpT accessory proteins.

The ATP-dependent caseinolytic protease (Clp) is an essential housekeeping enzyme in plant chloroplasts. It is by far the most complex of all known Clp proteases, with a proteolytic core consisting of multiple catalytic ClpP and noncatalytic ClpR subunits. It also includes a unique form of Clp protein of unknown function designated ClpT, two of which exist in the model species Arabidopsis thaliana. Inactivation of ClpT1 or ClpT2 significantly reduces the amount of Clp proteolytic core, whereas loss of both proves seedling lethal under autotrophic conditions. During assembly of the Clp proteolytic core, ClpT1 first binds to the P-ring (consisting of ClpP3-6 subunits) followed by ClpT2, and only then does the P-ring combine with the R-ring (ClpP1, ClpR1-4 subunits). Most of the ClpT proteins in chloroplasts exist in vivo as homodimers, which then apparently monomerize prior to association with the P-ring. Despite their relative abundance, however, the availability of both ClpT proteins is rate limiting for the core assembly, with the addition of recombinant ClpT1 and ClpT2 increasing core content up to fourfold. Overall, ClpT appears to regulate the assembly of the chloroplast Clp protease, revealing a new and sophisticated control mechanism on the activity of this vital protease in plants.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

Phenotypic and transcriptomic acclimation of the green microalga Raphidocelis subcapitata to high environmental levels of the herbicide diflufenican.

Herbicide pollution poses a worldwide threat to plants and freshwater ecosystems. However, the understanding of how organisms develop tolerance to these chemicals and the associated trade-off expenses are largely unknown. This study aims to investigate the physiological and transcriptional mechanisms underlying the acclimation of the green microalgal model species Raphidocelis subcapitata (Selenastraceae) towards the herbicide diflufenican, and the fitness costs associated with tolerance development. Algae were exposed for 12 weeks (corresponding to 100 generations) to diflufenican at the two environmental concentrations 10 and 310 ng/L. The monitoring of growth, pigment composition, and photosynthetic performance throughout the experiment revealed an initial dose-dependent stress phase (week 1) with an EC50 of 397 ng/L, followed by a time-dependent recovery phase during weeks 2 to 4. After week 4, R. subcapitata was acclimated to diflufenican exposure with a similar growth rate, content of carotenoids, and photosynthetic performance as the unexposed control algae. This acclimation state of the algae was explored in terms of tolerance acquisition, changes in the fatty acids composition, diflufenican removal rate, cell size, and changes in mRNA gene expression profile, revealing potential fitness costs associated with acclimation, such as up-regulation of genes related to cell division, structure, morphology, and reduction of cell size. Overall, this study demonstrates that R. subcapitata can quickly acclimate to environmental but toxic levels of diflufenican; however, the acclimation is associated with trade-off expenses that result in smaller cell size.

'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings.

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.

The chloroplast ATP-dependent Clp protease in vascular plants - new dimensions and future challenges.

The ATP-dependent Clp protease is by far the most intricate protease in chloroplasts of vascular plants. Structurally, it is particularly complex with a proteolytic core complex containing 11 distinct subunits along with three potential chaperone partners. The Clp protease is also essential for chloroplast development and overall plant viability. Over the past decade, many of the important characteristics of this crucial protease have been revealed in the model plant species Arabidopsis thaliana. Despite this, challenges still remain in fully resolving certain key features, in particular, how the assembly of this multisubunit protease is regulated, the full range of native protein substrates and how they are targeted for degradation and how this complicated enzyme might have developed from simpler bacterial forms. This article focuses upon the recent advances in revealing the details underlying these important features. It also take the opportunity to speculate upon many of these findings in the hope of stimulating further investigation.

Identification of new protein substrates for the chloroplast ATP-dependent Clp protease supports its constitutive role in Arabidopsis.

The ATP-dependent Clp protease in plant chloroplasts consists of a heterogeneous proteolytic core containing multiple ClpP and ClpR paralogues. In this study, we have examined in detail the only viable knockout mutant to date of one of these subunits in Arabidopsis thaliana, ClpR1. Loss of ClpR1 caused a slow-growth phenotype, with chlorotic leaves during early development that later partially recovered upon maturity. Analysis of the Clp proteolytic core in the clpR1 mutant (clpR1-1) revealed approx. 10% of the wild-type levels remaining, probably due to a relative increase in the closely related ClpR3 protein and its partial substitution of ClpR1 in the core complex. A proteomic approach using an in organello proteolytic assay revealed 19 new potential substrates for the chloroplast Clp protease. Many of these substrates were constitutive enzymes involved in different metabolic pathways, including photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis, whereas others function in housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling processes. In contrast, degradation of the stress-related chloroplast proteins Hsp21 (heat-shock protein 21) and lipoxygenase 2 was unaffected in the clpR1-1 line and thus not facilitated by the Clp protease. Overall, we show that the chloroplast Clp protease is principally a constitutive enzyme that degrades numerous stromal proteins, a feature that almost certainly underlies its vital importance for chloroplast function and plant viability.

Phenomics reveals a novel putative chloroplast fatty acid transporter in the marine diatom Skeletonema marinoi involved in temperature acclimation.

Diatoms are the dominant phytoplankton in temperate oceans and coastal regions and yet little is known about the genetic basis underpinning their global success. Here, we address this challenge by developing the first phenomic approach for a diatom, screening a collection of randomly mutagenized but identifiably tagged transformants. Based upon their tolerance to temperature extremes, several compromised mutants were identified revealing genes either stress related or encoding hypothetical proteins of unknown function. We reveal one of these hypothetical proteins is a novel putative chloroplast fatty acid transporter whose loss affects several fatty acids including the two omega-3, long-chain polyunsaturated fatty acids - eicosapentaenoic and docosahexaenoic acid, both of which have medical importance as dietary supplements and industrial significance in aquaculture and biofuels. This mutant phenotype not only provides new insights into the fatty acid biosynthetic pathways in diatoms but also highlights the future value of phenomics for revealing specific gene functions in these ecologically important phytoplankton.

Friends With Benefits: Exploring the Phycosphere of the Marine Diatom Skeletonema marinoi.

Marine diatoms are the dominant phytoplankton in the temperate oceans and coastal regions, contributing to global photosynthesis, biogeochemical cycling of key nutrients and minerals and aquatic food chains. Integral to the success of marine diatoms is a diverse array of bacterial species that closely interact within the diffusive boundary layer, or phycosphere, surrounding the diatom partner. Recently, we isolated seven distinct bacterial species from cultures of Skeletonema marinoi, a chain-forming, centric diatom that dominates the coastal regions of the temperate oceans. Genomes of all seven bacteria were sequenced revealing many unusual characteristics such as the existence of numerous plasmids of widely varying sizes. Here we have investigated the characteristics of the bacterial interactions with S. marinoi, demonstrating that several strains (Arenibacter algicola strain SMS7, Marinobacter salarius strain SMR5, Sphingorhabdus flavimaris strain SMR4y, Sulfitobacter pseudonitzschiae strain SMR1, Yoonia vestfoldensis strain SMR4r and Roseovarius mucosus strain SMR3) stimulate growth of the diatom partner. Testing of many different environmental factors including low iron concentration, high and low temperatures, and chemical signals showed variable effects on this growth enhancement by each bacterial species, with the most significant being light quality in which green and blue but not red light enhanced the stimulatory effect on S. marinoi growth by all bacteria. Several of the bacteria also inhibited growth of one or more of the other bacterial strains to different extents when mixed together. This study highlights the complex interactions between diatoms and their associated bacteria within the phycosphere, and that further studies are needed to resolve the underlying mechanisms for these relationships and how they might influence the global success of marine diatoms.

Whole Genome Sequence of Marinobacter salarius Strain SMR5, Shown to Promote Growth in its Diatom Host.

Attempts to obtain axenic cultures of the marine diatom Skeletonema marinoi often result in poor growth, indicating the importance of the microbiome to the growth of its host. In order to identify the precise roles played by these associated bacteria, individual strains were isolated, cultured and sequenced. We report the genome of one such strain - SMR5, isolated from a culture of S. marinoi strain R05AC sampled from top layer sediments of the Swedish west coast. Its genome of 4,630,160 bp consists of a circular chromosome and one circular plasmid, and 4,263 CDSs were inferred in the annotation. Comparison of 16S rRNA sequences and other markers, along with phylotaxonomic analysis, leads us to place strain SMR5 in the taxon Marinobacter salarius. Pathway analysis and previous experimental work suggest that this strain may produce a growth factor, as well as improve iron availability for its host via siderophores.

Complete Genome Sequence of the Diatom-Associated Bacterium Sphingorhabdus sp. Strain SMR4y.

The bacterial strain SMR4y belongs to the diverse microbiome of the marine diatom Skeletonema marinoi strain R05AC. After assembly of its genome, presented here, and subsequent analyses, we placed it in the genus Sphingorhabdus This strain has a 3,479,724-bp circular chromosome (with 3,340 coding sequences) and no known plasmids.

Skeletonema marinoi as a new genetic model for marine chain-forming diatoms.

Diatoms are ubiquitous primary producers in marine ecosystems and freshwater habitats. Due to their complex evolutionary history, much remains unknown about the specific gene functions in diatoms that underlie their broad ecological success. In this study, we have genetically transformed the centric diatom Skeletonema marinoi, a dominant phytoplankton species in temperate coastal regions. Transformation of S. marinoi is the first for a true chain-forming diatom, with the random genomic integration via nonhomologous recombination of a linear DNA construct expressing the resistance gene to the antibiotic zeocin. A set of molecular tools were developed for reliably identifying the genomic insertion site within each transformant, many of which disrupt recognizable genes and constitute null or knock-down mutations. We now propose S. marinoi as a new genetic model for marine diatoms, representing true chain-forming species that play a central role in global photosynthetic carbon sequestration and the biogeochemical cycling of silicates and various nutrients, as well as having potential biotechnological applications.

Genome Sequence of Arenibacter algicola Strain SMS7, Found in Association with the Marine Diatom Skeletonema marinoi.

Arenibacter algicola strain SMS7 was isolated from a culture of the marine diatom Skeletonema marinoi strain ST54, sampled from top-layer sediments in Kosterfjord, Sweden. Here, we present its 5,857,781-bp genome, consisting of a circular chromosome and one circular plasmid, in all containing 4,932 coding sequences.

Complete Genome Sequence of Novel Sulfitobacter pseudonitzschiae Strain SMR1, Isolated from a Culture of the Marine Diatom Skeletonema marinoi.

When studying diatoms, an important consideration is the role of associated bacteria in the diatom-microbiome holobiont. To that end, bacteria isolated from a culture of Skeletonema marinoi strain R05AC were sequenced, one of which being bacterial strain SMR1, presented here. The genome consists of a circular chromosome and seven circular plasmids, totalling 5,121,602 bp. After phylotaxonomic analysis and 16S rRNA sequence comparison, we place this strain in the taxon Sulfitobacter pseudonitzschiae on account of similarity to the type strain. The annotated genome suggests similar interactions between strain SMR1 and its host diatom as have been shown previously in diatom-associated Sulfitobacter, for example bacterial production of growth hormone for its host, and breakdown of diatom-derived DMSP by Sulfitobacter for use as a sulfur source.

Genome Sequence of Kordia sp. Strain SMS9 Identified in a Non-Axenic Culture of the Diatom Skeletonema marinoi.

Initial efforts to sequence the genome of the marine diatom Skeletonema marinoi were hampered by the presence of genetic material from bacteria, and there was sufficient material from some of these bacteria to enable the assembly of full chromosomes. Here, we report the genome of strain SMS9, one such bacterial species identified in a non-axenic culture of S. marinoi strain ST54. Its 5,482,391 bp circular chromosome contains 4,641 CDSs, and has a G+C content of 35.6%. Based on 16S rRNA comparison, phylotaxonomic analysis, and the genome similarity metrics dDDH and OrthoANI, we place this strain in the genus Kordia, and to the best of our knowledge, this is the first Kordia species to be initially described from European waters. As attempts to culture this strain have failed, however, the specifics of its relationship with S. marinoi are still uncertain.

Whole-Genome Sequence of the Novel Antarctobacter heliothermus Strain SMS3, Found in Association with the Marine Diatom Skeletonema marinoi.

As part of an ongoing investigation into the microbiome of the marine diatom Skeletonema marinoi, the bacterial strain SMS3 was isolated from a culture of S. marinoi strain ST54, which had been propagated from a sample of top layer marine sediments taken from the Swedish west coast. We present here the sequenced genome of this bacterium, which we place in the taxon Antarctobacter heliothermus, based on a phylotaxonomic analysis and its high 16S rRNA sequence similarity to the A. heliothermus type strain DSM 11445T. Its 5,331,190 bp genome consists of a circular chromosome and three circular plasmids, and contains 5,019 CDSs. Strain SMS3 contains a phosphatidylcholine synthase gene, as well as genes involved in DMSP degradation, both of which imply a potential symbiotic relationship with its host.

Complete Genome Sequence of Loktanella vestfoldensis Strain SMR4r, a Novel Strain Isolated from a Culture of the Chain-Forming Diatom Skeletonema marinoi.

We report here the genome sequence of Loktanella vestfoldensis strain SMR4r, isolated from the marine diatom Skeletonema marinoi strain RO5AC. Its 3,987,360-bp genome consists of a circular chromosome and two circular plasmids, one of which appears to be shared with an S. marinoi-associated Roseovarius species.

Genome Sequence of Roseovarius mucosus Strain SMR3, Isolated from a Culture of the Diatom Skeletonema marinoi.

We present the genome of Roseovarius mucosus strain SMR3, a marine bacterium isolated from the diatom Skeletonema marinoi strain RO5AC sampled from top layer sediments at 14 m depth. Its 4,381,426 bp genome consists of a circular chromosome and two circular plasmids and contains 4,178 coding sequences (CDSs).