Development of a two-color fluorescence in situ hybridization technique for species-level identification of human-infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.

Development of fluorescent in situ hybridization for Cryptosporidium detection reveals zoonotic and anthroponotic transmission of sporadic cryptosporidiosis in Sydney.

Cryptosporidium is the most common non-viral cause of diarrhea worldwide. Of the 5 described species that contribute to the majority of human infections, C. parvum is of major interest due to its zoonotic potential. A species-specific fluorescence in situ hybridisation probe was designed to the variable region in the small subunit of the 18S rRNA of C. parvum and labeled with Cy3. Probe specificity was validated against a panel of 7 other Cryptosporidium spp. before it was applied to 33 human faecal samples positive for cryptosporidiosis which were obtained during the period from 2006-2007. Results were compared to PCR-RFLP targeting the 18S rDNA. FISH results revealed that 19 of the 33 isolates analysed were identified as C. parvum. Correlation of PCR-RFLP and FISH was statistically significant (P<0.05), resulting in a calculated correlation coefficient of 0.994. In this study, species identification by FISH and PCR-RFLP provided preliminary evidence to support both anthroponotic and zoonotic transmission of sporadic cases of cryptosporidiosis in the Sydney basin. In conclusion, FISH using a C. parvum-specific probe provided an alternative tool for accurate identification of zoonotic Cryptosporidium which will be applied in the future to both epidemiological and outbreak investigations.

Applying fluorescence based technology to the recovery and isolation of Cryptosporidium and Giardia from industrial wastewater streams.

As increasing water shortages continue, water re-use is posing new challenges with treated wastewater becoming a significant source of non-potable water. Rapid detection strategies that target waterborne pathogens of concern to industry are gaining importance in the assessment of water quality. This study reports on the ability to recover spiked Cryptosporidium and Giardia from a variety of industrial wastewater streams of varied water quality. Incorporation of an internal quality control used commonly in finished water-enabled quantitative assessments of pathogen loads and we describe successful analysis of pre- and part-treated wastewater samples from four industrial sites. The method used combined calcium carbonate flocculation followed by flow cytometry and epifluorescence microscopy. Our focus will now aim at characterising the ambient parasites isolated from industrial wastewater with the objective of developing a suite of highly specific platform detection technologies targeted to industrial needs.

Secondary structure model for an unusual SSU rRNA from the extremely thermophilic bacterium strain AZ3 B.1.

The SSU rRNA gene of the extremely thermophilic bacterium strain AZ3 B.1, encodes an rRNA containing four large inserts. A secondary structure model has been constructed which predicts that the inserts form large stem loop structures with a common sequence motif at the base of the helices. To date, these structures have only been detected in related, thermophilic organisms.

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus.

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95,732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5'-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.

Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation.

The gene for a highly thermostable neutral proteinase (Npr) was isolated from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related species. The gene was sequenced and shown to be closely related to a neutral proteinase gene from Bacillus caldolyticus strain YP-T; the mature form of the enzyme differing by only a single amino acid. Enzyme samples were prepared from both the native organisms and also from recombinant Escherichia coli expressing the two npr genes. The proteinase from strain EA1 was shown to be significantly more thermostable than that from B. caldolyticus and that this difference is the result of a single amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The phylogenetic relationship of EA1 to other Bacilli is also described.

Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.

An Mr 29000 protein is essential for mini-F maintenance in E. coli.

Plasmids consisting of mini-F inserted into multicopy vectors were constructed. Derivatives of these hybrid replicons were isolated which contained the transposon Tn5. The polypeptides encoded by these plasmids were identified by Escherichia coli minicell analysis. We show that a previously unidentified polypeptide of 29000 Mr is encoded by the mini-F gene E between 45.1 and 46.2 F kb on the mini-F plasmid map, and that this coding sequence (E gene) is transcribed rightward. Hybrid plasmids carrying Tn5 inserted into the E gene are unable to replicate in a polA- strain. Hence the E protein is essential for mini-F replication. Mutations in the A and B genes of mini-F affect E gene expression, and the results suggest that E protein synthesis is stimulated by A protein.

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei.

The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xyn B in T. reesei was obtained after changing 20 codons.

A single emm gene-specific oligonucleotide probe does not recognise all members of the Streptococcus pyogenes M type 1.

Serological typing of the streptococcal M protein has recently been challenged by a number of unique molecular methodologies based on oligonucleotide recognition of allelic variations within the M protein (emm) gene. In these methods, stringent hybridization of an oligonucleotide probe to a polymerase chain reaction amplified emm gene is used as confirmation of specific M type identity. A sample of 17 isolates from 7 previously defined distinct genotypes were tested using a single M1 oligonucleotide probe. Isolates from only three of the genotypes hybridized with the probe. The results demonstrate that a single emm-specific oligonucleotide probe can not identify all members of M type 1, as defined by conventional serotyping using polyclonal antisera.

Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4.

A gene encoding a beta-mannanase (manA) has been cloned from an obligately anaerobic extreme thermophile, Caldicellulosiruptor strain Rt8B.4, which is most closely related to Caldicellulosiruptor saccharolyticus (formerly Caldocellum saccharolyticum). The gene codes for a multidomain enzyme with a C-terminal beta-mannanase domain which was amplified by the polymerase chain reaction and cloned into a temperature-inducible expression vector in Escherichia coli. Sequence comparisons have shown that the Man domain of Rt8B.4 ManA is related to a thermophilic Dictyoglomus mannanase and a mesophilic mannanase from a Bacillus species. It appears to be unrelated to the beta-mannanase domain of C. saccharolyticus, implying acquisition of the genes from unrelated sources by the two bacteria.

Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium.

A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.

Phylogeny and lipid composition of Thermonema lapsum, a thermophilic gliding bacterium.

1,490 nucleotides of the 16S rRNA gene of a Gram-negative, thermophilic and gliding bacterium, Thermonema lapsum, have been sequenced. Phylogenetic analysis indicates that T. lapsum is related to cytophaga-flavobacteria-bacteroides (CFB) and is confirmed by the identification signature nucleotides that define this group. Further phylogenetic analysis indicates that T. lapsum forms the deepest branch in the CFB group; this observation was confirmed by the identification of unique nucleotide and nucleotide pairs which separate T. lapsum from all other members of this group. The phospholipid fatty acid (PLFA) profile also confirmed that T. lapsum is related to the cytophaga-flavobacteria-bacteroides group and also to selected members of the genus Flexibacter; the PLFA profile is unique to T. lapsum.

Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system.

Effective biolistic transformation of intact conidia from the filamentous fungus Trichoderma reesei was achieved using the Bio-Rad Hepta Adaptor system with seven barrels for particle launch. Transformation frequencies of up to 39 colonies per microg of circular DNA and 37 colonies per microg of linear DNA were obtained at an optimal target distance of 3 cm and a helium pressure of 1350 psi. These values are about 3.5- to 6-fold higher than transformant yields reported earlier for T. reesei using the hygromycin phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin B as a selectable marker in combination with the PDS-1000/He single barrel system. High mitotic stability of the transformants (98-100%) was demonstrated. The Hepta Adaptor device allowing bombardment of seven lots of conidia in a single plate offers clear advantage in terms of transformant numbers over the single barrel system where target cells are restricted to the center of the plate.

Alteration of the pH optimum of a family 11 xylanase, XynB6 of Dictyoglomus thermophilum.

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions. One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases. Gene shuffling between badX and Dictyoglomus thermophilum xynB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6. Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH. With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX. The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 11 xylanases showing enhanced activity at alkaline pH's require multiple sequence adaptations across the protein.

The activity of family 11 xylanases at alkaline pH.

Xylanases have several industrial uses, particularly in baking, modification of animal feed and in pulp bleaching in the paper industry. Process conditions in kraft pulp bleaching generally favour an enzyme that is active at high pH values. The activities of several glycosyl hydrolase family 11 xylanases reported to be active under alkaline conditions were determined under optimal conditions and found to have optima in the pH 5-6 range. Only one enzyme tested, BadX, was shown to have an alkaline pH optimum. Significant activity at pH values higher than 8 appears often to be the result of excess enzyme added to the reaction mixtures so that substrate is limiting. The different nature of laboratory and industrial substrates needs to be taken into consideration in designing assay conditions. In some cases, significant differences were observed in pH profiles generated using a small-molecule substrate when compared to those generated using xylan. We conclude that small-molecule substrates are not a suitable proxy for determining the pH profiles of family 11 xylanases.