MlrA, an Essential Enzyme for Microcystins and Nodularin on First Step Biodegradation in Microcystin-Degrading Bacteria.

Microcystin-degrading bacteria first degrade microcystins by microcystinase A (MlrA) to cleave the cyclic structure of microcystins at the Adda-Arg site of microcystin-LR, microcystin-RR, and microcystin-YR, but the cleavage of the other microcystins was not clear. In our study, the microcystin-degrading bacterium Sphingopyxis sp. C-1 as wild type and that of mlrA-disrupting mutant, Sphingopyxis sp. CMS01 were used for microcystins biodegradation. The results showed MlrA degraded microcystin-LA, microcystin-LW, microcystin-LY, microcystin-LF, and nodularin. MlrA could cleave the Adda-L-amino acid site.

A device for assessing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI).

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high-pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H-leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep-sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep-sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep-sea biotic processes.

Dynamics of the prokaryotic and eukaryotic microbial community during a cyanobacterial bloom.

Toxic cyanobacterial blooms frequently develop in eutrophic freshwater bodies worldwide. Microcystis species produce microcystins (MCs) as a cyanotoxin. Certain bacteria that harbor the mlr gene cluster, especially mlrA, are capable of degrading MCs. However, MC-degrading bacteria may possess or lack mlr genes (mlr+ and mlr- genotypes, respectively). In this study, we investigated the genotype that predominantly contributes to biodegradation and cyanobacterial predator community structure with change in total MC concentration in an aquatic environment. The 2 genotypes coexisted but mlr+ predominated, as indicated by the negative correlation between mlrA gene copy abundance and total MC concentration. At the highest MC concentrations, predation pressure by Phyllopoda, Copepoda, and Monogononta (rotifers) was reduced; thus, MCs may be toxic to cyanobacterial predators. The results suggest that cooperation between MC-degrading bacteria and predators may reduce Microcystis abundance and MC concentration.

An overview of the accumulation of microcystins in aquatic ecosystems.

Cyanotoxins produced by toxic cyanobacteria pose a major, worldwide environmental threat to freshwater ecosystems. Microcystins (MCs) are considered to be the most hazardous groups. Indeed, some of the largest aquatic ecosystems on the earth are being contaminated with MCs. Questions have arisen regarding their transfer and bioaccumulation in natural environment. This review summarizes the present state of knowledge regarding toxic cyanobacteria and MCs, with a specific focus on their distribution in different components of aquatic ecosystems. Their accumulation in water columns, aquatic animals, plants, and sediments is summarized. MCs have been contaminating all areas of the aquatic ecosystems. Of these, the water column was the most contaminated with MCs and served as an intermediate transmission substation. Via this route, MCs could enter to other stations such as sediment, animals, aquatic and terrestrial plants. Therefore, the use of water contaminated with MCs may induce food chain contaminations with considerable health risks.

Phenotypic and genetic characterization of multidrug-resistant Staphylococcus aureus in the tropics of Southeast Asia.

Antibiotic resistance has become a major public health problem throughout the world. The presence of antibiotic-resistant bacteria such as Staphylococcus aureus and antibiotic resistance genes (ARGs) in hospital wastewater is a cause for great concern today. In this study, 276 Staph. aureus isolates were recovered from hospital wastewater samples in Malaysia. All of the isolates were screened for susceptibility to nine different classes of antibiotics: ampicillin, ciprofloxacin, gentamicin, kanamycin, erythromycin, vancomycin, trimethoprim and sulfamethoxazole, chloramphenicol, tetracycline and nalidixic acid. Screening tests showed that 100 % of Staph.aureus isolates exhibited resistance against kanamycin, vancomycin, trimethoprim and sulfamethoxazole and nalidixic acid. Additionally, 91, 87, 50, 43, 11 and 8.7 % of isolates showed resistance against erythromycin, gentamicin, ciprofloxacin, ampicillin, chloramphenicol and tetracycline, respectively. Based on these results, 100 % of isolates demonstrated multidrug-resistant (MDR) characteristics, displaying resistance against more than three classes of antibiotics. Of 276 isolates, nine exhibited resistance to more than nine classes of tested antibiotics; these were selected for antibiotic susceptibility testing and examined for the presence of conserved ARGs. Interestingly, a high percentage of the selected MDR Staph.aureus isolates did not contain conserved ARGs. These results indicate that non-conserved MDR gene elements may have already spread into the environment in the tropics of Southeast Asia, and unique resistance mechanisms against several antibiotics may have evolved due to stable, moderate temperatures that support growth of bacteria throughout the year.

Microcystin accumulation and biochemical responses in the edible clam Corbiculaleana P. exposed to cyanobacterial crude extract.

We investigated the accumulation and effects of cyanobacterial crude extract (CCE) containing microcystins (MCs) on the edible clam Corbiculaleana P. Toxic effects were evaluated through the activity of antioxidant and detoxification enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferases (GSTs) from gills, foot, mantle and remaining soft tissues. Clams were exposed to CCE containing 400µg MC-LReq/L for 10days and were then kept in toxin-free water for 5days. Clam accumulated MCs (up to 3.41±0.63µg/g dry weight (DW) of unbound MC and 0.31±0.013µg/g DW of covalently bound MC). Detoxification and antioxidant enzymes in different organs responded differently to CCE during the experiment. The activity of SOD, CAT, and GST in the gills and mantle increased in MC-treated clams. In contrast, CAT and GST activity was significantly inhibited in the foot and mostly only slightly changed in the remaining tissues. The responses of biotransformation, antioxidant enzyme activity to CCE and the fast elimination of MCs during depuration help to explain how the clam can survive for long periods (over a week) during the decay of toxic cyanobacterial blooms in nature.

Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C enhances generation of n-alkane-rich biofuels after pyrolysis.

We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH(4) production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Degradation of microcystins by an electrochemical oxidative electrode cell.

Microcystins (MCs), which are produced by cyanobacteria, are one of the most serious problems that threaten quality of drinking water and public health. In this study, an electrolysis cell with no electrolyte is demonstrated to degrade MCs (MC-RR, MC-YR and MC-LR) in both high and low concentrations. In addition, degradation of MCs was studied under different current densities. The results revealed that the electrolysis cell could degrade MCs successfully. It was observed that degradation of a single MC was faster than mixed types and statistical analysis revealed that the degradation rate of all the three MCs did not show much difference in mixed degradation. Analysis of hydroxyl radical concentration suggested a possible role of the hydroxyl radical in degradation of MCs. We propose that the electrolysis cell could be a promising treatment for effective removal of MCs in situ, especially in water purification plants where low amounts of salts (electrolytes) are present.

Draft Genome Sequences of Multidrug-Resistant Escherichia coli Strains Isolated from River Water in Malaysia.

Antimicrobial resistance has become a primary concern in clinical and public health. Escherichia coli is one of the bacteria that carries and disseminates antimicrobial resistance genes to the community. Here, we report the draft genome sequence of three multidrug-resistant E. coli strains that were isolated from river water in Malaysia.

Limited carbon cycling due to high-pressure effects on the deep-sea microbiome.

Deep-sea microbial communities are exposed to high-pressure conditions, which has a variable impact on prokaryotes depending on whether they are piezophilic (that is, pressure-loving), piezotolerant or piezosensitive. While it has been suggested that elevated pressures lead to higher community-level metabolic rates, the response of these deep-sea microbial communities to the high-pressure conditions of the deep sea is poorly understood. Based on microbial activity measurements in the major oceanic basins using an in situ microbial incubator, we show that the bulk heterotrophic activity of prokaryotic communities becomes increasingly inhibited at higher hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was about one-third of that measured in the same community at atmospheric pressure conditions. In the bathypelagic zone-between 1,000 and 4,000 m depth-~85% of the prokaryotic community was piezotolerant and ~5% of the prokaryotic community was piezophilic. Despite piezosensitive-like prokaryotes comprising only ~10% (mainly members of Bacteroidetes, Alteromonas) of the deep-sea prokaryotic community, the more than 100-fold metabolic activity increase of these piezosensitive prokaryotes upon depressurization leads to high apparent bulk metabolic activity. Overall, the heterotrophic prokaryotic activity in the deep sea is likely to be substantially lower than hitherto assumed, with major impacts on the oceanic carbon cycling.

Temperature affects growth, geosmin/2-methylisoborneol production, and gene expression in two cyanobacterial species.

Cyanobacterial blooms accompanied by taste and odor (T&O) compounds affect the recreational function and safe use of drinking water. Geosmin and 2-methylisoborneol (2-MIB) are the most common T&O compounds. In this study, we investigated the effect of temperature on geosmin and 2-MIB production in Dolichospermum smithii and Pseudanabaena foetida var. intermedia. More specifically, transcription of one geosmin synthase gene (geoA) and two 2-MIB synthase genes (mtf and mtc) was explored. Of the three temperatures (15, 25, and 35 °C) tested, the maximum Chl-a content was determined at 25 °C in both D. smithii and P. foetida var. intermedia. The maximum total geosmin concentration (19.82 µg/L) produced by D. smithii was detected at 25 °C. The total 2-MIB concentration (82.5 µg/L) produced by P. foetida var. intermedia was the highest at 35 °C. Besides, the lowest Chl-a content and minimum geosmin/2-MIB concentration were observed at 15 °C. There was a good positive correlation between geosmin/2-MIB concentration and Chl-a content. The expression levels of the geoA, mtf, and mtc genes at 15 °C were significantly higher than those at 25 and 35 °C. The transcription of the mtf and mtc genes in P. foetida var. intermedia was higher at 35 °C than at 25 °C. The results highlight unfavorable temperature can increase the potential of geosmin/2-MIB synthesis from the gene expression level in cyanobacteria. This study could provide basic knowledge of geosmin/2-MIB production by cyanobacteria for better understanding and management of T&O problems in drinking water.

Toxic cyanobacteria and microcystin dynamics in a tropical reservoir: assessing the influence of environmental variables.

Toxic cyanobacterial blooms (TCBs) have become a growing concern worldwide. The present study investigated the dynamic of toxic cyanobacteria and microcystin (MC) concentrations in the Tri An Reservoir (TAR), a tropical system in Vietnam, with quantitative real-time polymerase chain reaction (qPCR) and high-performance liquid chromatography (HPLC), respectively. The results of the qPCR quantification revealed that Microcystis was the dominant group and the primary MC producer in the TAR. Potentially toxigenic cyanobacteria varied from 1.2 × 104 to 1.58 × 107 cells/mL, and the mean proportion of toxic Microcystis to that of the total toxic cyanobacteria varied from 21 to 88%. Microcystin concentrations in raw water and sediment samples often peaked during June to October as blooms occurred and varied from 0.27 to 6.59 µg/L and from 1.79 to 544.9 ng/g in wet weight, respectively. The results of this study indicated that conditions favoring Microcystis proliferation lead to the selection of more toxic genotypes. Water temperature and light availability were not driving factor in the formation of TCBs in the TAR. However, the high loads of total nitrogen (TN), phosphate, and total phosphorus (TP) into the water via rainfall runoff in combination with a high total suspended solid (TSS) and decreased water level during the early months of the rainy seasons did lead to a shift in Microcystis blooms and higher proportions of toxic genotypes of Microcystis in the TAR. This research may provide more insight into the occurrence mechanism of TCBs in tropical waters. The strategy to control TCB problems in tropical regions should be focused on these limnological and hydrological parameters, in addition to a reduction in nitrogen and phosphorus loading.

Draft Genome Sequence of the Prazosin-Degrading Bacillus sp. Strain PR5, Isolated from a River Receiving Hospital and Urban Wastewater in Malaysia.

We report the complete genome sequence of Bacillus sp. strain PR5, isolated from a river receiving hospital and urban wastewater in Malaysia, which demonstrated a high capability for degrading prazosin. This genome sequence of 4,525,264 bp exhibited 41.5% GC content, 4,402 coding sequences, and 32 RNAs.

Effects of elevated nitrogen on the growth and geosmin productivity of Dolichospermum smithii.

Geosmin is one of the most common earthy-musty odor compounds, which is mainly produced by cyanobacteria in surface water. Nitrogen (N) is an important factor affecting the growth of cyanobacteria and its secondary metabolites production due to the eutrophication. In this study, we compared the effects of elevated N on the growth and geosmin productivity of Dolichospermum smithii NIES-824 (synonym Anabaena smithii NIES-824), aiming to better understand the mechanisms involved and give an important and fundamental knowledge to solve off-flavor problem. Results show that elevated N concentration promoted more chlorophyll a (Chl-a) production, whereas the geosmin synthesis decreased, revealing a possible competitive correlation between the Chl-a concentration and geosmin production of D. smithii NIES-824. The majority of geosmin (> 90%) was retained intracellularly during the 28 days of cultivation. The qRT-PCR analysis demonstrates that the expression level of the geosmin synthase gene (geoA) was constitutive and decreased at the higher N concentration during the exponential growth phase of cyanobacterial cells. Furthermore, the decrease of geoA expression during the decline phase suggested that geoA transcription was closely related to cell activity and isoprenoid productivity.

Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment.

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.

Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

Draft Genome Sequence of the Microcystin-Degrading Bacterium Novosphingobium sp. Strain MD-1.

This report describes the whole-genome sequence of a microcystin-degrading bacterium, Novosphingobium sp. strain MD-1, isolated from a lake in Japan. The Novosphingobium sp. strain MD-1 genome had a total length of 4,617,766 bp. Moreover, strain MD-1 showed a conserved microcystin-degrading gene cluster (mlrA to mlrF), similar to Sphingopyxis sp. strain C-1.

Coupling biogas recirculation with FeCl3 addition in anaerobic digestion system for simultaneous biogas upgrading, phosphorus conservation and sludge conditioning.

The high costs involved in sewage sludge treatment and disposal in wastewater treatment plants (WWTPs) not only bring about improper sludge disposal and thus environmental pollutions, but also limit the investment on construction of WWTPs, especially in rural areas or low-income regions. This comparative study examined the effect of biogas recirculation coupled with chemical addition in a semi-continuous anaerobic digester for sludge treatment, which was proven to achieve biogas upgrading, phosphorus conservation and sludge conditioning simultaneously, largely reducing the sludge treatment cost. Results show that FeCl3 addition coupling biogas recirculation can improve sludge dewaterability by 94% in comparison to 75% by equivalent MgCl2 addition, and 97% phosphorus in digestate can be conserved in solid with formation of vivianite-like crystals. Biogas recirculation can enhance CH4 yield and content by 13% and 11%, respectively, likely attributable to the increased relative abundances of both hydrogenotrophic Methanomicrobiales and acetoclastic Methanosarcinales.

Elucidation of prazosin biodegradation by isolated Bacillus spp. from the tropical environment.

Prazosin (PRZ), a drug used to treat hypertensive patients, is an emergent contaminant in water systems. PRZ is an alpha-adrenergic receptor blocker used to treat anxiety, and is believed to reach the environment through human excretion, irresponsible disposal of unused medicine, and waste products from manufacturing plants. The purpose of this research was to isolate and characterize potential microbes for PRZ biodegradation and to identify the degradation pathway. After screening, isolated strain STP3 showed a capability for PRZ degradation and was chosen for further analysis. Resting cell assays with PRZ were conducted to identify the intermediate metabolites formed from biodegradation by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) analysis. Two metabolites degraded from PRZ by STP3 were successfully found, and as these metabolites are derived from the main structure of PRZ, their presence proved PRZ degradation. Draft genome sequencing analysis of STP3 was performed to identify potential enzymes for PRZ biodegradation based on the metabolites found.

Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough.

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.