A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Genomic insights into Pseudoalteromonas sp. JSTW coping with petroleum-heavy metals combined pollution.

Worldwide marine compound contamination by petroleum products and heavy metals is a burgeoning environmental concern. Pseudoalteromonas, prevalently distributed in marine environment, has been proven to degrade petroleum and plays an essential role in the fate of oil pollution under the combined pollution. Nevertheless, the research on the reference genes is still incomplete. Therefore, this study aims to thoroughly investigate the reference genes represented by Pseudoalteromonas sp. JSTW via whole-genome sequencing. Next-generation sequencing technology unfolded a genome of 4,026,258 bp, database including Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were utilized to annotate the genes and metabolic pathways conferring to petroleum hydrocarbon degradation. The results show that common alkane and aromatic hydrocarbon degradation genes (alkB, ligB, yqhD, and ladA), chemotaxis gene (MCP, cheA, cheB, pcaY, and pcaR), heavy-metal resistance, and biofilm genes (σ54, merC, pcoA, copB, etc.) were observed in whole-genome sequence (WGS) of JSTW, which indicated that strain JSTW could potentially cope with combined pollution. The degradation efficiency of naphthalene in 60 h by JSTW was 99% without Cu2+ and 67% with 400 mg L-1 Cu2+ . Comparative genome analysis revealed that genomes of Pseudoalteromonas lipolytica strain LEMB 39 and Pseudoalteromonas donghaensis strain HJ51 shared similarity with strain JSTW, suggesting they are also the potential degradater of petroleum hydrocarbons under combined pollution. Therefore, this study provides a WGS annotation and reveals the mechanism of response to combined pollution of Pseudoalteromonas sp. JSTW.

Biodegradation of crude oil using self-immobilized hydrocarbonoclastic deep sea bacterial consortium.

Hydrocarbonoclastic bacterial consortium that utilizes crude oil as carbon and energy source was isolated from marine sediment collected at a depth of 2100 m. Molecular characterization by 16S rRNA gene sequences confirmed that these isolates as Oceanobacillus sp., Nesiotobacter sp., Ruegeria sp., Photobacterium sp., Enterobacter sp., Haererehalobacter sp., Exiguobacterium sp., Acinetobacter sp. and Pseudoalteromonas sp. Self-immobilized consortium degraded more than 85% of total hydrocarbons after 10 days of incubation with 1% (v/v) of crude oil and 0.05% (v/v) of Tween 80 (non-ionic surfactant) at 28 ±â€¯2 °C. The addition of nitrogen and phosphorus sources separately i.e. 0.1% (v/v) of CO (NH2)2 or K2HPO4 enhanced the hydrocarbon utilization percentage. The pathways of microbial degradation of hydrocarbons were confirmed by FTIR, GC-MS, 1H and 13C NMR spectroscopy analyses. These results demonstrated a novel approach using hydrocarbonoclastic self-immobilized deep sea bacterial consortium for eco-friendly bioremediation.

Expression, purification and characterization of halophilic protease Pph_Pro1 cloned from Pseudoalteromonas phenolica.

In this study, protease Pph_Pro1 from Pseudoalteromonas phenolica, possessing extracellular proteolytic activity and salt tolerance, was investigated for cloning, expression, and purification purposes. Through optimization, it was determined that optimum soluble recombinant expression was achieved when Pph_Pro1 was co-expressed with the pTf16 vector chaperone in LB medium supplemented with CaCl2. Pph_Pro1 was purified using osmotic shock and immobilized metal-affinity chromatography (IMAC). Isolated Pph_Pro1 activity was measured as 0.44 U/mg using casein as a substrate. Interestingly, Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostable properties. Furthermore, it was resistant to several hydrophilic and hydrophobic organic solvents. Substrate specificity and kinetic values such as Km and Vmax were determined with casein, bovine serum albumin (BSA), and algal waste protein as substrates, indicating that the Pph_Pro1 protease enzyme had a greater affinity for casein. Based on the remarkable characteristics of this Pph_Pro1 protease enzyme, it can potentially be utilized in many biotechnological industries.

Changes in bacterial community metabolism and composition during the degradation of dissolved organic matter from the jellyfish Aurelia aurita in a Mediterranean coastal lagoon.

Spatial increases and temporal shifts in outbreaks of gelatinous plankton have been observed over the past several decades in many estuarine and coastal ecosystems. The effects of these blooms on marine ecosystem functioning and particularly on the dynamics of the heterotrophic bacteria are still unclear. The response of the bacterial community from a Mediterranean coastal lagoon to the addition of dissolved organic matter (DOM) from the jellyfish Aurelia aurita, corresponding to an enrichment of dissolved organic carbon (DOC) by 1.4, was assessed for 22 days in microcosms (8 l). The high bioavailability of this material led to (i) a rapid mineralization of the DOC and dissolved organic nitrogen from the jellyfish and (ii) the accumulation of high concentrations of ammonium and orthophosphate in the water column. DOM from jellyfish greatly stimulated heterotrophic prokaryotic production and respiration rates during the first 2 days; then, these activities showed a continuous decay until reaching those measured in the control microcosms (lagoon water only) at the end of the experiment. Bacterial growth efficiency remained below 20%, indicating that most of the DOM was respired and a minor part was channeled to biomass production. Changes in bacterial diversity were assessed by tag pyrosequencing of partial bacterial 16S rRNA genes, DNA fingerprints, and a cultivation approach. While bacterial diversity in control microcosms showed little changes during the experiment, the addition of DOM from the jellyfish induced a rapid growth of Pseudoalteromonas and Vibrio species that were isolated. After 9 days, the bacterial community was dominated by Bacteroidetes, which appeared more adapted to metabolize high-molecular-weight DOM. At the end of the experiment, the bacterial community shifted toward a higher proportion of Alphaproteobacteria. Resilience of the bacterial community after the addition of DOM from the jellyfish was higher for metabolic functions than diversity, suggesting that jellyfish blooms can induce durable changes in the bacterial community structure in coastal lagoons.

Biodegradation of complex hydrocarbons in spent engine oil by novel bacterial consortium isolated from deep sea sediment.

Complex hydrocarbon and aromatic compounds degrading marine bacterial strains were isolated from deep sea sediment after enrichment on spent engine (SE) oil. Phenotypic characterization and phylogenetic analysis of 16S rRNA gene sequences showed the isolates were related to members of the Pseudoalteromonas sp., Ruegeria sp., Exiguobacterium sp. and Acinetobacter sp. Biodegradation using 1% (v/v) SE oil with individual and mixed strains showed the efficacy of SE oil utilization within a short retention time. The addition of non-ionic surfactant 0.05% (v/v) Tween 80 as emulsifying agent enhanced the solubility of hydrocarbons and renders them more accessible for biodegradation. The degradation of several compounds and the metabolites formed during the microbial oxidation process were confirmed by Fourier transform infrared spectroscopy and Gas chromatography-mass spectrometry analyses. The potential of this consortium to biodegrade SE oil with and without emulsifying agent provides possible application in bioremediation of oil contaminated marine environment.

Towards industrially feasible treatment of potato starch processing waste by mixed cultures.

The present study aimed at reducing the pollution of the waste generated by the potato starch industry to the environment and transform the potato pulp and wastewater into single-cell protein (SCP) to be used as animal feed. The chemical oxygen demand of the wastewater was reduced from 26,700 to 9,100 mg/L by batch fermentation with mixed cultures in an aerated 10-L fermenter. The SCP products, with a crude protein content of 46.09 % (higher than soybean meal), were found palatable and safe for mice. During the treatment process, the microbial community was analyzed using the terminal restriction fragment length polymorphism for bacterial 16S rRNA genes. The results of the analysis suggested that Curacaobacter/Pseudoalteromonas and Paenibacillus/Bacillus were the main microorganisms in treating potato starch processing wastes. The 150-m(3)-scale fermentation demonstrated a potential for treatment in industrial applications. Fermentation of potato pulp and wastewater without adding an extra nitrogen source was a novel approach in treating the potato starch processing waste.

Identification of compounds with bioactivity against the nematode Caenorhabditis elegans by a screen based on the functional genomics of the marine bacterium Pseudoalteromonas tunicata D2.

Marine bacteria are a rich, yet underexplored, resource of compounds with inhibitory bioactivity against a range of eukaryotic target organisms. Identification of those inhibitors, however, requires a culturable or genetically tractable producer strain, a prerequisite that is not often fulfilled. This study describes a novel functional genomic screen that is based on expression of inhibitors in a heterogeneous recombinant host (i.e., Escherichia coli). Functional libraries were screened by selective grazing by the nematode Caenorhabditis elegans, in a simple, rapid, high-throughput manner. We applied our approach to discover inhibitors of C. elegans produced by the marine bacterium Pseudoalteromonas tunicata D2, a model organism for exploring a range of antagonistic activities between bacteria and eukaryotes and a known producer of several toxic compounds. Expression of P. tunicata DNA in E. coli and grazing selection by the nematode Caenorhabditis elegans identified two clones, with slow- and fast-killing modes of action. Genomic analysis of the slow-killing clone revealed that the activity was due to a small molecule, tambjamine, while the fast-killing activity involved a gene encoding for a novel protein. Microscopic analysis showed substantial colonization of the intestinal lumen, or rapid death of the nematode without colonization, for the two activities, respectively. The novel functional genomic screen presented here therefore detects new eukaryotic inhibitors with different chemical structures, kinetics, and predicted modes of actions.

[A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2].

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca2+ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 x 10(4)-2 x 10(5) infectious centers per microgram of PM2 DNA was developed.