Species-Specific Secondary Metabolite Production in Marine Actinomycetes of the Genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S–23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.     

Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

Chemotaxonomy of <Emphasis Type="Italic">Pochonia</Emphasis> and other conidial fungi with <Emphasis Type="Italic">Verticillium</Emphasis>-like anamorphs

Pochonins are antiviral and antiparasitic resorcylic acid lactones (RAL) structurally related to monorden. They were found in the invertebrate-associated fungus Pochonia chlamydosporia. Their production and distribution was studied by means of High Performance Liquid Chromatography with UV-visual and mass spectrometric detection (HPLC-UV/Vis and HPLCMS) in cultures of Pochonia species and further conidial fungi with Verticillium-like anamorphs that had until recently been included in Verticillium sect. Prostrata. The results support the recent generic segregation by Gams, Zare and co-workers because pochonins were found to occur exclusively in species of the genus Pochonia. With few exceptions, the production of RAL appeared to be a rather constant feature in cultures of P. chlamydosporia from around the world. According to preliminary results, secondary metabolite profiles in strains of allied genera such as Lecanicillium, Haptocillium and Rotiferophthora are different from those encountered in Pochonia. The alkaloid pseurotin A was found as main metabolite in several of the P. chlamydosporia isolates examined. As inferred from HPLC profiling data, strains of P. suchlasporia clustered into at least three chemotypes. The ex-type strain of P. suchlasporia var. catenata produced monorden, while several other strains produced metabolites whose HPLC-UV and HPLC-MS characteristics were similar to the mycotoxins, aurovertin B and citreoviridin A. Yet different metabolites were detected in a third chemotype of P. suchlasporia. Differences in secondary metabolite profiles were also found in two strains of P. bulbillosa. While the ex-type strain was found devoid of all aforementioned compounds, CBS 247.68 contained the aurovertin-related metabolites detected in part of the P. suchlasporia isolates. The sequence of the ITS nrDNA of CBS 247.68 was different from that of the type strain but identical to the sequences of P. suchlasporia var. catenata. Several strains of the latter variety showed identical sequences, despite considerable variations in their HPLC metabolite profiles. Minisatellite PCR fingerprinting was found useful to segregate Pochonia at species and strain level, pointing toward the existence of further, cryptic species. The possible chemotaxonomical importance and ecological functions of secondary metabolites in these fungi is discussed.     

Characterization and evolution of secondary metabolites in Brazilian Vernonieae (Asteraceae) assessed by LC‐MS fingerprinting

Tribe Vernonieae are well represented in Brazil, mainly by subtribe Lychnophorinae comprising mostly perennial plants that inhabit arid lands, where species have developed a number of adaptations with recognized ecological protective functions, including secondary metabolite diversification. Recent phylogenetic studies indicate that the subtribe is monophyletic, but questions regarding lineage relationships have yet to be resolved. Phytochemical investigations have also been conducted recently and provide information on the secondary metabolite chemistry of Lychnophorinae. Chemotaxonomic studies have also been carried out. However, these phytochemical investigations are fragmentary and non‐standardized. Therefore, in this study, 15 species representing the major lineages of the subtribe were selected for phytochemical investigation and reconstruction of the ancestral states of their secondary metabolites. The main secondary metabolites of these species were detected by UHPLC‐UV‐MS in different types of extract, showing the presence of trans‐cinnamic acid derivatives, flavonoids, polyacetylenes and sesquiterpene lactones. The ancestral states of these secondary metabolites were reconstructed by parsimony and indicate the occurrence of 12 putative chemical synapomorphies. In this study, we present for the first time phytochemical and evolutionary studies based on the reconstruction of the ancestral chemical character states on a phylogenetic tree of Lychnophorinae.     

Soft coral Sarcophyton (Cnidaria: Anthozoa: Octocorallia) species diversity and chemotypes

Research on the soft coral genus Sarcophyton extends over a wide range of fields, including marine natural products and the isolation of a number of cembranoid diterpenes. However, it is still unknown how soft corals produce this diverse array of metabolites, and the relationship between soft coral diversity and cembranoid diterpene production is not clear. In order to understand this relationship, we examined Sarcophyton specimens from Okinawa, Japan, by utilizing three methods: morphological examination of sclerites, chemotype identification, and phylogenetic examination of both Sarcophyton (utilizing mitochondrial protein-coding genes MutS homolog: msh1) and their endosymbiotic Symbiodinium spp. (utilizing nuclear internal transcribed spacer of ribosomal DNA: ITS- rDNA). Chemotypes, molecular phylogenetic clades, and sclerites of Sarcophyton trocheliophorum specimens formed a clear and distinct group, but the relationships between chemotypes, molecular phylogenetic clade types and sclerites of the most common species, Sarcophyton glaucum, was not clear. S. glaucum was divided into four clades. A characteristic chemotype was observed within one phylogenetic clade of S. glaucum. Identities of symbiotic algae Symbiodinium spp. had no apparent relation to chemotypes of Sarcophyton spp. This study demonstrates that the complex results observed for S. glaucum are due to the incomplete and complex taxonomy of this species group. Our novel method of identification should help contribute to classification and taxonomic reassessment of this diverse soft coral genus.     

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.     

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.     

Identification of NephrotoxicPenicillium Species from Cereal Grains

A system is described for identifying grain-inhabiting nephrotoxicPenicillium spp. based on their colony characters on Czapek yeast extract agar, yeast extract sucrose agar, and malt extract agar media, and their secondary metabolite profiles on thin layer chromatography plates. Using this system, the identity of 11Penicillium species, or their chemotypes, producing nephrotoxic metabolites could be confirmed. The species areP. verrucosum chemotype I, P.verrucosum chemotype II,P. expansum, P. citrinum, P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. viridicatum, P. cyclopium, and P. melanoconldlum. Other non-nephrotoxicPenicillium species present on stored grains were separated from nephrotoxic species by their colony characters and metabolite profiles.     

Phylogenetic and Chemical Diversity of a Hybrid-Isoprenoid-Producing Streptomycete Lineage

Streptomyces species dedicate a large portion of their genomes to secondary metabolite biosynthesis. A diverse and largely marine-derived lineage within this genus has been designated MAR4 and identified as a prolific source of hybrid isoprenoid (HI) secondary metabolites. These terpenoid-containing compounds are common in nature but rarely observed as bacterial secondary metabolites. To assess the phylogenetic diversity of the MAR4 lineage, complementary culture-based and culture-independent techniques were applied to marine sediment samples collected off the Channel Islands, CA. The results, including those from an analysis of publically available sequence data and strains isolated as part of prior studies, placed 40 new strains in the MAR4 clade, of which 32 originated from marine sources. When combined with sequences cloned from environmental DNA, 28 MAR4 operational taxonomic units (0.01% genetic distance) were identified. Of these, 82% consisted exclusively of either cloned sequences or cultured strains, supporting the complementarity of these two approaches. Chemical analyses of diverse MAR4 strains revealed the production of five different HI structure classes. All 21 MAR4 strains tested produced at least one HI class, with most strains producing from two to four classes. The two major clades within the MAR4 lineage displayed distinct patterns in the structural classes and the number and amount of HIs produced, suggesting a relationship between taxonomy and secondary metabolite production. The production of HI secondary metabolites appears to be a phenotypic trait of the MAR4 lineage, which represents an emerging model with which to study the ecology and evolution of HI biosynthesis.     

PRODUCTION OF SECONDARY METABOLITES BY FILAMENTOUS TROPICAL MARINE CYANOBACTERIA: ECOLOGICAL FUNCTIONS OF THE COMPOUNDS

Cyanobacterial blooms are becoming more common in many reef habitats. The broadly acting feeding deterrent compound ypaoamide, produced by a mixed cyanobacterial assemblage, has been linked to bloom formation and mass fish die-offs ( Siganus argenteus and Siganus spinus ) in Guam. Specific metabolites produced by Lyngbya majuscula Gomont act as both feeding attractants to the specialist herbivore Stylocheilus longicauda , and as effective feeding deterrents to generalist fishes. Two-dimensional TLC (2D-TLC) analysis of cyanobacterial crude extracts was used to select chemically distinct populations (chemotypes) of bloom-forming filamentous cyanobacteria for chemical and ecological evaluation. Crude extracts produced by different species, chemotypes, and chemically distinct Micronesian marine cyanobacterial assemblages deter feeding activity of generalist reef herbivores. The ecological function of cyanobacterial secondary metabolites, especially as related to diversity of compound production and the relationship of metabolite production to bloom formation is discussed.     

Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences

Characteristics and structures of mt SSU rDNA were investigated for the phylogenetic study of Ganoderma. Phylogenetic information was concentrated mostly in the V1, V4, V5, V6 and V9 variable domains, but informative sites in conserved domains also significantly contributed in resolving phylogenetic relationships between Ganoderma groups. Secondary structure information of variable domains was found to be a useful marker in delineation of phylogenetic groups. Strains of Ganoderma species used in this study were divided into six monophyletic groups. Ganoderma colossus made a distinct basal lineage from other Ganoderma species and Tomophagus, created for G. colosuss, appeared to be a valid genus. Ganoderma applanatum and G. lobatum classified in subgenus Elfvingia made a monophyletic group. Ganoderma tsugae from North America and G. valesiacum from Europe, both living on conifers, were closely related. Ganoderma oregonense and strains labeled G. lucidum from Europe and Canada were grouped with G. tsugae and G. valesiacum. Strains labeled G. lucidum living on hardwoods from the United States and Taiwan were grouped with G. resinaceum, G. pfeifferi and G. subamboinense var. laevisporum, and they all produced chlamydospores. Two strains labeled G. lucidum and three strains labeled G. resinaceum from America were concluded to be conspecific. Strains labeled G. lucidum from Korea and Japan were monophyletic and were distinguished from strains labeled G. lucidum from Europe and North America. Host relationships and the presence of chlamydospores in culture proved to be important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.     

Cultural Growth Patterns and Incompatibility Reactions in Diaporthe and Phomopsis Populations

Comparative studies were carried out with cultures isolated from Phomopsis condiomata and/or Diaporthe ascomata found on naturally-infected living stems or on overwintered stem fragments of Arctium lappa L., Helianthus annuus L., Glycine max (L.) Merr., and Xanthium italicum Moretti, growing in or nearby the same fields in Vojvodina. Results underscored the heterogeneity of the Phomopsis anamorphs in the D. arctii (Lasch) Trav, complex, Intra-specific vegetative incompatibility groups were distinguished among Phomopsis and/or Diaporthe populations from A. lappa. More than one phytopathogenic Phomopsis and/or Diaporthe species were identified on X. italicum. P. longicolla Hobbs was isolated for the first time in Yugoslavia from soybean stem debris. The holomorph Diaporthe (Phomopsis) helianthi Muntanola- Cvetkovic, Mihaljcevic et Petrov showed distinguishing characteristics with respect to other congeneric fungi.     

Molecular phylogeny and taxonomic revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and description of Oogamochlamys gen. nov. and Lobochlamys gen. nov

The genus Chlamydomonas (including Chloromonas) is one of the largest green algal genera comprising more than 600 species. To initiate a comprehensive analysis of the phylogeny and systematics of the genus, we determined nuclear-encoded SSU rRNA sequences from 32 strains of Chlamydomonas, Chloromonas and Chlorogonium with emphasis on oogamous taxa and related strains, and incorporated these into global molecular phylogenetic analyses of 132 strains of Chlorophyceae. In addition, we studied the morphology and reproduction of oogamous and related strains by light microscopy. We recognize and designate 18 monophyletic lineages (clades) within the Chlorophyceae, 11 of which are confined to the CW (basal bodies displaced clockwise) subgroup. The majority of clades recognized within the Chlorophyceae do not correspond to any of the traditional classification systems, which are still largely based on the organization level. Strains assigned to Chlamydomonas and Chloromonas were found in seven different clades confirming the polyphyly of the two genera as presently conceived. To initiate the taxonomic revision of Chlamydomonas, C. reinhardtii is proposed as the conserved type of the genus. In consequence, species in clades other than the clade containing C. reinhardtii must be transferred to other genera, a process initiated in this contribution. The oogamous strains studied represent a monophyletic lineage, which is described as Oogamochlamys gen. nov. comprising three species (O. gigantea, O. zimbabwiensis and O. ettlii spec. nov.). The sister clade to Oogamochlamys consists of isogamous strains characterized by chloroplasts with incisions and is described as Lobochlamys gen. nov. with two species (L. culleus and L. segnis). Another clade is characterized by asteroid or perforated, parietal chloroplasts and contains the type species of Chloromonas (C. reticulata). Thus, the polyphyletic Chloromonas (traditionally defined as “Chlamydomonas without pyrenoids”) can be legitimized as a monophyletic genus by restriction to this clade and is here emended on the basis of chloroplast characters (the clade contains strains with or without pyrenoids thus rejecting the character “absence of pyrenoids”).     

Chemotaxonomy of Trichoderma spp. Using mass spectrometry-based metabolite profiling

In this study, seven Trichoderma species (33 strains) were classified using secondary metabolite profile-based chemotaxonomy. Secondary metabolites were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) and multivariate statistical methods. T. longibrachiatum and T. virens were independently clustered based on both internal transcribed spacer (ITS) sequence and secondary metabolite analyses. T. harzianum formed three subclusters in the ITS-based phylogenetic tree and two subclusters in the metabolitebased dendrogram. In contrast, T. koningii and T. atroviride strains were mixed in one cluster in the phylogenetic tree, whereas T. koningii was grouped in a different subcluster from T. atroviride and T. hamatum in the chemotaxonomic tree. Partial least-squares discriminant analysis (PLS-DA) was applied to determine which metabolites were responsible for the clustering patterns observed for the different Trichoderma strains. The metabolites were hetelidic acid, sorbicillinol, trichodermanone C, giocladic acid, bisorbicillinol, and three unidentified compounds in the comparison of T. virens and T. longibrachiatum; harzianic acid, demethylharzianic acid, homoharzianic acid, and three unidentified compounds in T. harzianum I and II; and koninginin B, E, and D, and six unidentified compounds in T. koningii and T. atroviride. The results of this study demonstrate that secondary metabolite profiling-based chemotaxonomy has distinct advantages relative to ITSbased classification, since it identified new Trichoderma clusters that were not found using the latter approach.     

Identification and phylogenetic analysis of Arsenophonus- and Photorhabdus-type bacteria from adult Hippoboscidae and Streblidae (Hippoboscoidea)

This is the first report of Arsenophonus- and Photorhabdus-type bacteria from Streblidae (bat flies) and Hippoboscidae (louse flies, keds). Strains were detected by means of polymerase chain reaction of 16S rDNA, and phylogenetic analysis determined the relationship of the obtained sequences to previously reported sequences in GenBank. Phylogenetic analysis by means of maximum parsimony revealed that all isolated Arsenophonus spp. 16S rDNA sequences formed a monophyletic sub-clade within other insect Arsenophonus spp., while the Photorhabdus spp. sequences are part of a monophyletic clade including Photorhabdus spp., Xenorhabdus spp. and Proteus spp.