Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans.

AIMS: Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. METHODS AND RESULTS: A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. SIGNIFICANCE AND IMPACT OF STUDY: Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

Diversity and antimicrobial activities of microbes from two Irish marine sponges, Suberites carnosus and Leucosolenia sp.

AIMS: To evaluate the diversity and antimicrobial activity of bacteria from the marine sponges Suberites carnosus and Leucosolenia sp. METHODS AND RESULTS: Two hundred and thirty-seven bacteria were isolated from the sponges S. carnosus (Demospongiae) and Leucosolenia sp. (Calcarea). Isolates from the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were obtained. Isolates of the genus Pseudovibrio were dominant among the bacteria from S. carnosus, whereas Pseudoalteromonas and Vibrio were the dominant genera isolated from Leucosolenia sp. Approximately 50% of the isolates from S. carnosus displayed antibacterial activity, and c. 15% of the isolates from Leucosolenia sp. demonstrated activity against the test fungal strains. The antibacterial activity observed was mostly from Pseudovibrio and Spongiobacter isolates, while the majority of the antifungal activity was observed from the Pseudoalteromonas, Bacillus and Vibrio isolates. CONCLUSIONS: Both sponges possess a diverse range of bioactive and potentially novel bacteria. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggest that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which cultured bacterial isolates from the marine sponges S. carnosus and a Leucosolenia sp. have been evaluated for their antibacterial activity. The high percentage of antibacterial isolates from S. carnosus and of antifungal isolates from Leucosolenia sp. suggests that these two sponges may be good sources for potentially novel marine natural products.

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems.

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function-based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic-based screens.

Diversity and antimicrobial activity of Pseudovibrio spp. from Irish marine sponges.

AIMS: To evaluate the diversity and antimicrobial activity present among Pseudovibrio spp. isolated from marine sponges. METHODS AND RESULTS: Seventy-three bacterial isolates from the marine sponges Polymastia boletiformis, Axinella dissimilis and Haliclona simulans were identified as Pseudovibrio spp. using phylogenetic analysis of 16S rRNA gene sequences. Genetic diversity among these isolates was estimated using random amplification of polymorphic DNA (RAPD), and 33 RAPD types were identified among the 73 Pseudovibrio isolates. These Pseudovibrio spp. were assayed for the production of compounds with antimicrobial activity against various clinically relevant pathogens. Sixty-two (85%) of the isolates showed activity against at least one of the pathogens tested, including Escherichia coli, Salmonella enterica serotype Typhimurium, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium difficile. PCR screens of the Pseudovibrio isolates also revealed the presence of potential antibiotic-producing polyketide synthase genes. CONCLUSIONS: Marine sponges harbour a diverse population of Pseudovibrio spp., the majority of which demonstrate antimicrobial activity. The identification of several different antimicrobial activity spectra suggests that the Pseudovibrio isolates may produce a suite of antimicrobial compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which an extended population of Pseudovibrio isolates from marine sponges has been analysed and establishes the little-studied Pseudovibrio as a potentially important genus in the search for antimicrobial compounds of clinical relevance.

Deposition of substituted apatites with anticolonizing properties onto titanium surfaces using a novel blasting process.

A series of doped apatites have been deposited onto titanium (V) substrates using a novel ambient temperature blasting process. The potential of these deposited doped apatites as non-colonizing osteoconductive coatings has been evaluated in vitro. XPS, EDX, and gravimetric analysis demonstrated that a high degree of coating incorporation was observed for each material. The modified surfaces were found to produce osteoblast proliferation comparable to, or better than, a hydroxyapatite finish. Promising levels of initial microbial inhibition were observed from the Sr- and Ag-doped surfaces, with the strontium showing prolonged ability to reduce bacteria numbers over a 30-day period. Ion elution profiles have been characterized and linked to the microbial response and based on the results obtained, mechanisms of kill have been suggested. In this study, the direct contact of coated substrate surfaces with microbes was observed to be a significant contributing factor to the antimicrobial performance and the anticolonizing activity. The silver substituted apatite was observed to out-perform both the SrA and ZnA in terms of biofilm inhibition.

Endoglucanase activities and growth of marine-derived fungi isolated from the sponge Haliclona simulans.

AIMS: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge-derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. METHODS AND RESULTS: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate-based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5-dinitrosalicylic acid (DNSA)-based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. CONCLUSIONS: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine-derived fungi produced high endoglucanase activities that were especially active at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Marine-derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus.

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein.

Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10.

A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

The role of indigenous yeasts in traditional Irish cider fermentations.

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.

Real-time PCR analysis of carbon catabolite repression of cellobiose dehydrogenase gene transcription in Trametes versicolor.

Cellobiose dehydrogenase production in Trametes versicolor is repressed when additional carbon sources, such as glucose, maltose, galactose, arabinose, and xylose, are added to the fungal cultures growing on cellulose. Real-time quantitative reverse transcription-polymerase chain reaction has been used to demonstrate that the addition of galactose, arabinose, and xylose results in 19-, 92-, and 114-fold reductions, respectively, in cdh transcript levels 96 h post-addition. Glucose exhibits the greatest repressive effect, resulting in a 3400-fold decrease in cdh transcript levels.

Parameters affecting biological phosphate removal from wastewaters.

This paper reviews some of the key wastewater composition parameters, which influence the biological removal of phosphate from wastewaters, such as COD content, volatile fatty acid (VFA) content, cation concentration, phosphorus load, pH and food to microorganism ratio. The discussion also focuses on operational parameters affecting successful nutrient removal in wastewater treatment plants, such as temperature, sludge quality, sludge settlement, dissolved oxygen (DO) concentration, anaerobic P-release and secondary P-release. The aim of this review is to compile an updated document for researchers and operators of biological nutrient removal (BNR) systems. In addition, the article will provide a good foundation for readers with no prior knowledge of the process.

A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus.

Ochratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1.4 kb clone shared 28-35 % identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.

The use of amplified flanking region-PCR in the isolation of laccase promoter sequences from the edible fungus Pleurotus sajor-caju.

AIMS: To determine the regulation of laccase isozyme gene transcription in Pleurotus sajor-caju in response to different aromatic inducers and physiological parameters. METHODS AND RESULTS: The promoter regions for each of four different laccase isozymes were cloned from P. sajor-caju, using amplified flanking region-PCR (AFR-PCR). Sequences stretching 724, 214, 840 and 1740 bp upstream from the predicted start codons for lac1, lac2, lac3 and lac4, respectively, were cloned in each case and analysed for the presence of putative transcriptional response elements. A number of putative response elements including metal response elements, xenobiotic response elements and antioxidant response elements appear to be present. In addition putative consensus sequences such as those for the binding of AP1, AP2, creA and NIT2 transcription factors, which are involved in nitrogen and carbon regulation in different fungi, are also present in the promoter regions of some of the isozymes. CONCLUSIONS: These elements may be involved in the transcriptional regulation of laccase gene expression in P. sajor-caju. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of a number of putative transcriptional response elements in the promoter regions of different isozyme genes indicates a potential role for these sites in regulating laccase gene transcription in P. sajor-caju. In addition this work demonstrates the potential usefulness of AFR-PCR as a technique to clone fungal DNA sequences located upstream from known sequences.

Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid.

AIMS: To develop a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect viable Candida krusei contaminations and examine the potential role of the citrate synthase (cs1) gene in adaptation to acetic acid. METHODS AND RESULTS: Fruit juice artificially contaminated with C. krusei cells was heat treated to inactivate the yeast cells, after which the improved ability of the RT-PCR over the PCR assay, through the amplification of the cs1 gene, to differentiate viable contaminations was shown. The sensitivity of the detection assay was 6 x 104 CFU ml-1. RT-PCR and densitometric analysis of the cs1 gene throughout the process of adaptation to acetic acid highlighted a potential role for the gene in the yeast's adaptive response. CONCLUSIONS: The RT-PCR assay through the targeting of the cs1 gene proved to be a specific, sensitive and direct method for the identification of a C. krusei contamination in a food environment. The cs1 gene was shown to play a potential role in the adaptation of the culture to the weak-acid preservative acetic acid. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: The development of a direct, sensitive and specific identification assay for C. krusei from a food environment and understanding the mechanism employed in adapting to a preservative challenge, represent important tools to the food industry in attempting to limit spoilage by this important food spoilage yeast.

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host.

The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris. The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris.

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 microM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation.