Using Molecular Networking for Microbial Secondary Metabolite Bioprospecting.

The oceans represent an understudied resource for the isolation of bacteria with the potential to produce novel secondary metabolites. In particular, actinomyces are well known to produce chemically diverse metabolites with a wide range of biological activities. This study characterised spore-forming bacteria from both Scottish and Antarctic sediments to assess the influence of isolation location on secondary metabolite production. Due to the selective isolation method used, all 85 isolates belonged to the phyla Firmicutes and Actinobacteria, with the majority of isolates belonging to the genera Bacillus and Streptomyces. Based on morphology, thirty-eight isolates were chosen for chemical investigation. Molecular networking based on chemical profiles (HR-MS/MS) of fermentation extracts was used to compare complex metabolite extracts. The results revealed 40% and 42% of parent ions were produced by Antarctic and Scottish isolated bacteria, respectively, and only 8% of networked metabolites were shared between these locations, implying a high degree of biogeographic influence upon secondary metabolite production. The resulting molecular network contained over 3500 parent ions with a mass range of m/z 149-2558 illustrating the wealth of metabolites produced. Furthermore, seven fermentation extracts showed bioactivity against epithelial colon adenocarcinoma cells, demonstrating the potential for the discovery of novel bioactive compounds from these understudied locations.

Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631.

Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (Tm = 99.82°C, ΔHcal = 4.58 × 10(4) cal mol(-1)). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.

Chondroitin Lyase from a Marine Arthrobacter sp. MAT3885 for the Production of Chondroitin Sulfate Disaccharides.

Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5-7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.

Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa.

Moritella viscosa causes winter ulcer disease in salmonids. The aim of the present work was to isolate and partially characterise an extracellular peptidase from M. viscosa, and to study its role in virulence. The peptidase, termed MvP1, was a 38-kDa metallopeptidase produced in late exponential growth. The optimum temperature for MvP1 was 40 degrees C, but the enzyme was active over a broad range of temperatures. MvP1 was non-lethal to salmon at concentrations up to 0.22microg/g fish, but extracellular products were lethal to salmon. MvP1 degraded casein, gelatin and collagen from lumpfish skin. It caused considerable tissue necrosis and hemorrhages at the site of injection, and affected cell-cell adhesions in EPC and BF-2 cell lines, but was not highly cytotoxic. The peptidase partially degraded fish IgM heavy chain but was non-hemolytic. The mvp1 gene was sequenced and encoded a 734-aa polypeptide containing a signal sequence, an N-terminal propeptide, a mature peptidase domain and a C-terminal propeptide. The MvP1 propeptide undergoes both N-terminal and C-terminal processing and different C-terminal processing results in the formation of several active isoforms of the mature peptidase. The catalytic domain showed highest sequence similarity with several vibriolysins (EC 3.4.24.25) originating from Pseudoalteromonas strains, showing up to 80% aa identity. The results indicate that MvP1 is a previously unknown vibriolysin that might affect M. viscosa virulence by aiding in the invasion and dissemination of the bacterium in its host, by causing tissue destruction.

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland.

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus.

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADeltasp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.

Characterization of a 5'-polynucleotide kinase/3'-phosphatase from bacteriophage RM378.

A polynucleotide kinase from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus was identified, expressed, and purified. This polynucleotide kinase was demonstrated to have a 5'-kinase domain as well as a 3'-phosphohydrolase domain. The RM378 polynucleotide kinase had limited sequence similarity to the 5'-kinase domain of the T4 bacteriophage polynucleotide kinase, but apparent homology was not evident within the 3'-phosphohydrolase domain. The domain order of RM378 polynucleotide kinase was reversed relative to that of the T4 polynucleotide kinase. The RM378 phosphohydrolase domain displayed some sequence similarity with the bacterial poly(A) polymerase family, including an HD motif characteristic of the diverse superfamily of metal-dependent HD phosphohydrolases. The RM378 polynucleotide kinase was biochemically characterized and shown to possess 5'-kinase activity on RNA and single- and double-stranded DNA at elevated temperatures. It also showed phosphohydrolase activity on 2':3'-cyclic adenosine monophosphate. This description of the RM378 polynucleotide kinase, along with the recently described RM378 RNA ligase, suggests that the RM378 bacteriophage has to counter a similar anti-phage mechanism in R. marinus as the one that the T4 phage has to counter in Escherichia coli.

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.