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Chemotype: A Chemically Distinct Entity with Implications for Medicine, Agriculture, and Industry



A chemotype (sometimes chemovar) is a chemically distinct entity in a plant or microorganism, with differences in the composition of the secondary metabolites. Minor genetic and epigenetic changes with little or no effect on morphology or anatomy may produce large changes in the chemical phenotype. Chemotypes are often defined by the most abundant chemical produced by that individual and the concept has been useful in work done by chemical ecologists and natural product chemists. With respect to plant biology, the term "chemotype" was first coined by Rolf Santesson and his son Johan in 1968, defined as, "...chemically characterized parts of a population of morphologically indistinguishable individuals."[1]




chemotype



In microbiology, the term "chemoform" or "chemovar" is preferred in the 1990 edition of the International Code of Nomenclature of Bacteria (ICNB), the former referring to the chemical constitution of an organism and the latter meaning "production or amount of production of a particular chemical." Terms with the suffix -type are discouraged so as to avoid confusion with type specimens.[2] The terms chemotype and chemovar were originally introduced to the ICNB in a proposed revision to one of the nomenclatural rules dealing with infrasubspecific taxonomic subdivisions at the 1962 meeting of the International Microbiological Congress in Montreal. The proposed change argued that nomenclatural regulation of these ranks, such as serotype and morphotype, is necessary to avoid confusion. In proposed recommendation 8a(7), it was asked that "authorization be given for the use of the terms chemovar and chemotype," defining the terms as being "used to designate an infrasubspecific subdivision to include infrasubspecific forms or strains characterized by the production of some chemical not normally produced by the type strain of the species." The change to the Code was approved in August 1962 by the Judicial Commission of the International Committee of Bacteriological Nomenclature at the VIII International Microbiological Congress in Montreal.[3]


A good example of a plant with many polymorphic chemotypes is Thymus vulgaris. While largely indistinguishable in appearance, specimens of T. vulgaris may be assigned to one of seven different chemotypes, depending on whether the dominant component of the essential oil is thymol, carvacrol, linalool, geraniol, sabinene hydrate (thuyanol), α-terpineol, or eucalyptol. Such chemotypes may be indicated as Thymus vulgaris ct. thymol (red thyme), or Thymus vulgaris ct. geraniol (sweet thyme), etc. Such an indication has no taxonomic standing.[1]


Because chemotypes are defined only by the most abundant secondary metabolite, they may have little practical meaning as a group of organisms sharing the same trait. Individuals of one chemotype may have vastly different chemical profiles, varying in the abundance of kind of the next most abundant chemical. This means two individuals of the same chemotype could have different impacts on herbivores, pollinators, or resistance to pests. A study by Ken Keefover-Ring and colleagues in 2008 cautioned that, "...this can be a very qualitative assessment of an individual's chemical profile, under which may be hiding significant chemical diversity."[1]


The fungal genus Stachybotrys produces several diverse toxins that affect human health. Its strains comprise two mutually-exclusive toxin chemotypes, one producing satratoxins, which are a subclass of trichothecenes, and the other producing the less-toxic atranones. To determine the genetic basis for chemotype-specific differences in toxin production, the genomes of four Stachybotrys strains were sequenced and assembled de novo. Two of these strains produce atranones and two produce satratoxins.


Comparative analysis of these four 35-Mbp genomes revealed several chemotype-specific gene clusters that are predicted to make secondary metabolites. The largest, which was named the core atranone cluster, encodes 14 proteins that may suffice to produce all observed atranone compounds via reactions that include an unusual Baeyer-Villiger oxidation. Satratoxins are suggested to be made by products of multiple gene clusters that encode 21 proteins in all, including polyketide synthases, acetyltransferases, and other enzymes expected to modify the trichothecene skeleton. One such satratoxin chemotype-specific cluster is adjacent to the core trichothecene cluster, which has diverged from those of other trichothecene producers to contain a unique polyketide synthase.


The two toxin chemotypes of Stachybotrys. Both atranones and satratoxins are terpenoid secondary metabolites thought to derive from the primary metabolite farnesyl pyrophosphate (FPP). Box colors indicate each class of molecule and its specific secondary metabolite precursors: blue for atranones, green for simple trichothecenes, and pink for macrocyclic trichothecenes, which include satratoxins. Atranones are diterpenoids thought to originate from cyclization of geranylgeranyl pyrophosphate to form dolabellane, which has an eleven-membered ring [11]. Shown are the structures of all atranones solved by Hinkley et al.[11], as well as types of enzymes capable of catalyzing the two postulated reactions in the pathway. Trichothecenes are sesquiterpenoids that are products of FPP cyclization. The pathway of trichodermol biosynthesis from FPP is known experimentally [12, 13], but there are no experimental data regarding biosynthesis pathways of satratoxins or other trichodermol derivatives. Shown is a conceptual pathway adapted from [14] and references therein. It integrates results from several trichothecene producers. Enzymes shown have been functionally characterized from Fusarium (Tri5) or Trichoderma (Tri4 and Tri11). Trichodiol is shown to represent several intermediates that undergo both enzymatic hydroxylation and spontaneous rearrangement to form trichodermol, which is the first molecule shown that contains the trichothecene skeleton, i.e., the tricyclic ring 12,13-epoxytrichothec-9-ene (EPT). In Fusarium, trichodermol is not observed. Instead, the pathway after trichodiol diverges into a series of products substituted at C-3 of EPT. There are two known trichoverrols (A and B) and two known trichoverrins (A and B), but the respective pairs differ only in the stereochemistry of the C-4 side chain. The satratoxin F/G skeleton is shown as representative of satratoxins, and roridin E as representative of roridins. Omitted for brevity are the verrucarins (double arrow between roridins and satratoxins).


To determine the genetic basis for the two chemotypes of Stachybotrys and to compare Stachybotrys to other trichothecene toxin producers including Fusarium and Trichoderma, the genomes of four cultured Stachybotrys strains were sequenced and assembled de novo. Two of these strains make atranones, and the other two make satratoxins. Some global properties of these genomes are reported, most notably their richness of polyketide synthase (PKS) genes. The core trichothecene cluster (CTC) of Stachybotrys is presented and shown to diverge significantly from the CTCs of other trichothecene producers, with a genomic context that appears to be chemotype-specific. Finally, comparative methods are used to support the hypothesis that the toxin chemotype in Stachybotrys may arise from the presence of strain-specific secondary metabolite biosynthesis gene clusters, including three satratoxin chemotype-specific clusters and a novel 35-kbp locus that has been named the core atranone cluster (CAC).


Conceptual and ortholog-based maximum likelihood phylogeny of Stachybotrys and other fungi. A. The conceptual phylogeny shows the toxin chemotypes of the four sequenced Stachybotrys strains in relation to other trichothecene-producing fungi of order Hypocreales. S. cerevisiae is only distantly related to Hypocreales and is shown for context. Topology adapted from [18]. B. Phylogeny was constructed from alignment of 2,177 proper protein orthologs identified by OrthoMCL. Scale bar shows number of substitutions per site. All branches have 100% support.


The core trichothecene clusters of Stachybotrys, Trichoderma, and F. graminearum, and satratoxin chemotype-specific clusters SC1, SC2, and SC3 of Stachybotrys. A. The core trichothecene cluster (CTC). For all genomes an arrow indicates a gene and its transcriptional sense. The core trichothecene clusters of Stachybotrys are shown in the green box, and the adjacent satratoxin cluster SC3 is shown in the pink box. The other genes that are shown outside the boxes lack similarity to known trichothecene synthesis genes, so they are assumed to be in flanking regions outside these two clusters. A black, dotted arrow indicates that a scaffold extends to include other genes beyond the region shown, whereas lack of such an arrow indicates a scaffold border. The color indicates orthology with respect to Trichoderma and F. graminearum trichothecene clusters (shown in the gray, dotted box). TRI18, which is a paralog of TRI3, is colored as TRI3, but the arrow is dotted. Note that Trichoderma TRI5 is known to exist outside of the CTC [12]. The ruler at the top indicates length in kbp. Trichoderma and F. graminearum CTCs were redrawn from prior work [12, 29]. B. The satratoxin-specific clusters are shown in the pink boxes. The other genes shown are chemotype-independent. Other figure conventions follow those described for the CTC.


Most Stachybotrys paralogs of Fusarium and Trichoderma trichothecene synthesis genes are found within the Stachybotrys CTC. However, also identified are two Stachybotrys loci outside of the CTC that contain paralogs of Stachybotrys CTC genes. First, there is the satratoxin chemotype-specific cluster SC2 (Figure 4B), which contains paralogs of TRI3 and TRI4. Second, the assembly of strain 40293 includes a small scaffold (not shown) that contains only two genes. They have been named TRI19 and TRI20 and are paralogs of TRI5 and TRI6, respectively. Stachybotrys orthologs of other known Fusarium trichothecene biosynthesis genes have not been identified in these assemblies. In particular, the trichothecene exporter TRI12, which is present in the CTCs of both Fusarium and Trichoderma[12], is absent in Stachybotrys. 2ff7e9595c


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