A Global Approach to Estimating the Abundance and Duplication of Polyketide Synthase Domains in Dinoflagellates.

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known.

The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates.

BACKGROUND: Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. RESULTS: The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. CONCLUSIONS: Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.

Translating the message: Karlodinium veneficum possesses an expanded toolkit of protein translation initiation factors.

Dinoflagellates are unusual eukaryotes with large genomes and a reduced role for transcriptional regulation compared to other eukaryotes. The mRNA in dinoflagellates is trans-spliced with a 5'-spliced-leader sequence, yielding a 22-nucleotide 5'-sequence with a methylated nucleotide cap. Since the control of gene expression is primarily post-transcriptional, this study focuses on mRNA recruitment as a means for regulating gene expression and specifically on the diversity of eIF4E family members. Three novel clades related to the cap binding initiation factor eIF4E have been recognized in alveolates that are distinct from the three metazoan classes of eIF4E. We have analyzed the characteristics of five of the fourteen eIF4E family members from Karlodinium veneficum, four from clade 1 and one from clade 2. Members of each clade all bear the distinctive features of a cap-binding protein. We examined their ability to interact with the cap analogue, m7GTP using an in vitro bead-binding assay. We show that recombinant eIF4E-1 family members are able to bind the cap analogue m7GTP, but eIF4E-2b binds poorly. Overall, the eIF4E-1 family members may be serving as general cap-binding translation initiation factors, while the eIF4E-2 (and perhaps eIF4E-3) family members may serve a regulatory role in gene expression.

Altered expression of isoniazid-regulated genes in drug-treated dormant Mycobacterium tuberculosis.

OBJECTIVES: Despite having potent activity against actively replicating Mycobacterium tuberculosis, isoniazid has very limited activity against dormant bacilli. In order to investigate the lack of bactericidal activity of this drug under conditions leading to mycobacterial dormancy, we studied the transcriptional pattern of M. tuberculosis in different physiological states following exposure to isoniazid. METHODS: Global gene expression analysis was used to study M. tuberculosis treated with isoniazid in dormancy models of nutrient depletion and progressive hypoxia in vitro, as well as in an in vivo hollow fibre model of dormancy. Mycobacterial expression of the drug's putative transcriptional signature was investigated by RT-PCR in each dormancy model, and during the early and chronic phases of infection in the mouse aerosol model. Transcriptional responses were correlated with the bactericidal activity of isoniazid in the respective models. RESULTS: A small group of genes directly relevant to the mechanism of action of isoniazid was confirmed to constitute a transcriptional signature of the drug, as differential regulation of these genes was abrogated in an isoniazid-resistant, katG-deficient M. tuberculosis strain following isoniazid exposure. Isoniazid-induced expression of this transcriptional signature was abolished in dormant bacilli which had acquired phenotypic tolerance to isoniazid, regardless of the specific conditions responsible for the induction of the dormancy phenotype. Quantitative RT-PCR revealed that expression of isoniazid-regulated genes (IRGs) is dramatically altered under conditions of nutrient depletion and progressive hypoxia in vitro. Although these IRGs are highly induced following drug exposure early in infection in the mouse hollow fibre and aerosol models, correlating with potent bactericidal activity of the drug, their expression levels are markedly diminished during late-stage infection in these two models, coinciding with the greatly reduced bactericidal activity of isoniazid against these organisms. CONCLUSIONS: The reduced susceptibility of bacilli to the bactericidal drug isoniazid, as well as lack of expression of IRGs upon exposure to the drug, may be defining features of M. tuberculosis dormancy.

Mycobacterium tuberculosis SigF regulates genes encoding cell wall-associated proteins and directly regulates the transcriptional regulatory gene phoY1.

Mycobacterium tuberculosis SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions, M. tuberculosis sigF is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in M. tuberculosis entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a sigF-deficient mutant has altered cell membrane properties. Using an M. tuberculosis sigF deletion mutant, we show here that sigF is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress sigF, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of sigF resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following sigF induction was Rv3301c (phoY1), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates phoY1, whose proposed promoter sequence is GGATTG-N(16)-GGGTAT.