Peter L. Bergquist

Comparative morphology and molecular phylogenetic analysis of three new species of the genus Karenia (Dinophyceae) from New Zealand

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake & Kominami ex Oda) G. Hansen & Moestrup, Karenia brevis (Davis) G. Hansen & Moestrup, and Karenia brevisulcata (Chang) G. Hansen & Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater & Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.

Prospecting for novel lipase genes using PCR

A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described. The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design. Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA. This lipase showed less than 20% similarity with other known lipases at the amino acid level. The study also revealed that distantly related members of the alpha/beta hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.

A randomisation test of the null hypothesis that two cladograms are sample estimates of a parametric phylogenetic tree

Abstract Cladograms for the same group of taxa derived using different datasets often agree extensively but are seldom identical. This disagreement may be due to the fact that cladograms are sampling estimates of the true phylogeny and as a result may differ only because of sampling error. A protocol is proposed to test the null hypothesis that two trees estimate the true or parametric phylogeny and are no more different than would be expected due to sampling error. In the event that the null hypothesis is rejected, the datasets are pruned to remove potentially confounding information, and the test of the null hypothesis is repeated. If the null hypothesis cannot be rejected, a method for combining the cladistic information from both datasets is proposed that takes account of the variability of the cladistic structure. The procedure is illustrated using morphological and molecular data from genera of the sponge Order Hadromerida (Porifera: Demospongiae).

CelA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum

Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources. Cellulase together on its genome. The gene for one of the cellulases (celA) was isolated on a λ genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids. To date, it is the largest cellulase gene sequenced. The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers). The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1,4-β-d-glucanases. The carboxylterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose. In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.

Phylogeny of twenty Thermus isolates constructed from 16S rRNA gene sequence data

The sequences of the 16S rRNA genes of 20 Thermus isolates were determined to a high fidelity by using automated DNA sequencing and fluorescent-dye-labelled primers. The strains tested included members of the three validly named Thermus species and representatives of major taxonomic clusters defined previously for this genus. The parsimony method was used to reconstruct the phylogeny of the strains from the aligned sequences, and a bootstrap analysis revealed a number of well-supported clades. Our results are not consistent with groupings inferred from numerical taxonomy data but support the conjecture that the genus Thermus contains more species than the three currently recognized species.

Detection and enumeration of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) using rRNA-targeted oligonucleotide probes

Abstract Effective management of fish aquaculture stocks to mitigate the impact of fish-killing phytoplankton requires intensive spatial and temporal sampling to identify and quantify potentially harmful species. so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of large-subunit rRNA (LSU rRNA)–targeted oligonucleotide probes as tools to aid in the detection and enumeration of the fragile, fish-killing species Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae). Oligonucleotides directed toward H. akashiwo and F. japonica were evaluated using fluorescent in situ hybridization (FISH). Probes that labelled those species well in the FISH format were then incorporated into a sandwich hybridization assay (SHA). SHAs were successfully developed for both H. akashiwo and F. japonica. Batch culture experiments showed that the response of the SHA using a constant number of H. akashiwo and F. japonica cells harvested in exponential vs stationary phase of growth varied by a factor of approximately two. Preliminary field trials indicate that the SHA appears to be a faster, more cost-effective, and easier-to-use method for detecting and estimating the abundance of H. akashiwo and F. japonica than is FISH or conventional light microscopy. This is particularly true when large numbers of samples need to be processed routinely and rapidly, and when the organism in question is fragile and subject to lysis or morphological distortion if chemically preserved prior to microscopical observation.

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei

Aims: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei.

Sequence structure and expression of a cloned β-glucosidase gene from an extreme thermophile

SummaryThe gene for a β-glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of Mr 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the β-glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.

Solid-binding peptides: smart tools for nanobiotechnology

Over the past decade, solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to the surfaces of a diverse range of solid materials including metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers, and minerals. They can direct the assembly and functionalisation of materials, and have the ability to mediate the synthesis and construction of nanoparticles and complex nanostructures. As the availability of newly synthesised nanomaterials expands rapidly, so too do the potential applications for SBPs.

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (kcat/KM) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (kcat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (Ki) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.