David J. Midgley

Fungal identification using a Bayesian classifier and the Warcup training set of internal transcribed spacer sequences

Fungi are key organisms in many ecological processes and communities. Rapid and low cost surveys of the fungal members of a community can be undertaken by isolating and sequencing a taxonomically informative genomic region, such as the ITS (internal transcribed spacer), from DNA extracted from a metagenomic sample, and then classifying these sequences to determine which organisms are present. This paper announces the availability of the Warcup ITS training set and shows how it can be used with the Ribosomal Database Project (RDP) Bayesian Classifier to rapidly and accurately identify fungi using ITS sequences. The classifications can be down to species level and use conventional literature-based mycological nomenclature and taxonomic assignments.

Microbial biodiversity in a Malaysian oil field and a systematic comparison with oil reservoirs worldwide

Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined. A total of 1,056 16S rRNA gene clones were screened from each location by amplified ribosomal DNA restriction analysis. All samples were dominated by clones affiliated with Marinobacter, some novel Deferribacteraceae genera and various clones allied to the Methanococci. In addition, either Marinobacterium- or Pseudomonas-like operational taxonomic units were detected from either compartment. A systematic comparison with the existing pertinent studies was undertaken by analysing the microbial amplicons detected and the PCR primers used. The analyses demonstrated that bacterial communities were site specific, while Archaea co-occurred more frequently. Amplicons related to Marinobacter, Marinobacterium and Pseudomonas were detected in a number of the studies examined, suggesting they may be ubiquitous members in oil reservoirs. Further analysis of primers used in those studies suggested that most primer pairs had fairly broad but low matches across the bacterial and archaeal domains, while a minority had selective matches to certain taxa or low matches to all the microbial taxa tested. Thus, it indicated that primers may play an important role in determining which taxa would be detected.

Draft Genome Sequence of Clostridium sporogenes PA 3679, the Common Nontoxigenic Surrogate for Proteolytic Clostridium botulinum

Clostridium sporogenes PA 3679 is widely used as a nontoxigenic surrogate for proteolytic strains of Clostridium botulinum in the derivation and validation of thermal processes in food. Here we report the draft assembly and annotation of the C. sporogenes PA 3679 genome. Preliminary analysis demonstrates a high degree of relatedness between C. sporogenes PA 3679 and sequenced strains of proteolytic C. botulinum.

Reanalysis and Simulation Suggest a Phylogenetic Microarray Does Not Accurately Profile Microbial Communities

The second generation (G2) PhyloChip is designed to detect over 8700 bacteria and archaeal and has been used over 50 publications and conference presentations. Many of those publications reveal that the PhyloChip measures of species richness greatly exceed statistical estimates of richness based on other methods. An examination of probes downloaded from Greengenes suggested that the system may have the potential to distort the observed community structure. This may be due to the sharing of probes by taxa; more than 21% of the taxa in that downloaded data have no unique probes. In-silico simulations using these data showed that a population of 64 taxa representing a typical anaerobic subterranean community returned 96 different taxa, including 15 families incorrectly called present and 19 families incorrectly called absent. A study of nasal and oropharyngeal microbial communities by Lemon et al (2010) found some 1325 taxa using the G2 PhyloChip, however, about 950 of these taxa have, in the downloaded data, no unique probes and cannot be definitively called present. Finally, data from Brodie et al (2007), when re-examined, indicate that the abundance of the majority of detected taxa, are highly correlated with one another, suggesting that many probe sets do not act independently. Based on our analyses of downloaded data, we conclude that outputs from the G2 PhyloChip should be treated with some caution, and that the presence of taxa represented solely by non-unique probes be independently verified.

Aspergillus hancockii sp. Nov., a biosynthetically talented fungus endemic to southeastern Australian soils

Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.

Draft Genome Sequence of Methanobacterium sp. Maddingley, Reconstructed from Metagenomic Sequencing of a Methanogenic Microbial Consortium Enriched from Coal-Seam Gas Formation Water

ABSTRACT The draft genome of Methanobacterium sp. Maddingley was reconstructed from metagenomic sequencing of a methanogenic microbial consortium enriched from coal-seam gas formation water. It is a hydrogenotrophic methanogen predicted to grow using hydrogen and carbon dioxide.

Genomic and chemical insights into biosurfactant production by the mangrove-derived strain Bacillus safensis CCMA-560

Many Bacillus species can produce biosurfactant, although most of the studies on lipopeptide production by this genus have been focused on Bacillus subtilis. Surfactants are broadly used in pharmaceutical, food and petroleum industry, and biological surfactant shows some advantages over the chemical surfactants, such as less toxicity, production from renewable, cheaper feedstocks and development of novel recombinant hyperproducer strains. This study is aimed to unveil the biosurfactant metabolic pathway and chemical composition in Bacillus safensis strain CCMA-560. The whole genome of the CCMA-560 strain was previously sequenced, and with the aid of bioinformatics tools, its biosurfactant metabolic pathway was compared to other pathways of closely related species. Fourier transform infrared (FTIR) and high-resolution TOF mass spectrometry (MS) were used to characterize the biosurfactant molecule. B. safensis CCMA-560 metabolic pathway is similar to other Bacillus species; however, some differences in amino acid incorporation were observed, and chemical analyses corroborated the genetic results. The strain CCMA-560 harbours two genes flanked by srfAC and srfAD not present in other Bacillus spp., which can be involved in the production of the analogue gramicidin. FTIR and MS showed that B. safensis CCMA-560 produces a mixture of at least four lipopeptides with seven amino acids incorporated and a fatty acid chain with 14 carbons, which makes this molecule similar to the biosurfactant of Bacillus pumilus, namely, pumilacidin. This is the first report on the biosurfactant production by B. safensis, encompassing the investigation of the metabolic pathway and chemical characterization of the biosurfactant molecule.

Draft Genome Sequence of Bacillus pumilus CCMA-560, Isolated from an Oil-Contaminated Mangrove Swamp.

ABSTRACT Bacillus pumilus strain CCMA-560 was isolated from an oil-contaminated mangrove swamp and was shown to produce biosurfactants. The strain appears to be capable of degrading some plant cell wall-related compounds, including hemicelluose and pectin. Genes for biopolymer export and polysaccharide intercellular adhesin synthesis were also annotated.

Morphological and molecular data for Australian Hebeloma species do not support the generic status of Anamika

The first collection of a macrofungal agaric species, with morphological features similar to already described Anamika species, has been found in association with animal bones in north Queensland, Australia. This species also shares features with several, commonly occurring and previously described Australian Hebeloma species. An integrated morphological and molecular study has resulted in the conclusion that all Anamika species belong in Hebeloma. As a result, already described species of Anamika are recombined as H. indicum (K.A. Thomas, Peintner, M.M. Moser and Manim.) B.J. Rees & Orlovich, H. angustilamellatum (Zhu L. Yang and Z.W. Ge) B.J. Rees & Orlovich and H. lactariolens (Clémençon and Hongo) B.J. Rees & Orlovich. A. phylogenetic tree based on ribosomal ITS sequences examines the relationship of these species with other Hebeloma species from both hemispheres. Four new species, Hebeloma youngii B.J. Rees, H. nothofagetorum B.J. Rees, H. subvictoriense B.J. Rees, H. lacteocoffeatum B.J. Rees, and one form, H. aminophilum f. hygrosarx B.J. Rees, are described as new from Australia.

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

ABSTRACT We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.