mlrA Gene Research Articles

Accumulation of microcystins (MCs), bacterial community composition and mlrA gene abundance in shrimp culture ponds

Accumulation of microcystins (MCs), bacterial community composition and mlrA gene abundance in shrimp culture ponds

Whole-cell and Pure Enzyme-catalyzed Degradation of Microcystins: A Comparative Study of the Efficacy of Heterologously Expressed mlrA Gene

The increasing occurrence of cyanobacterial blooms, driven by factors such as water eutrophication and climate warming, has raised concerns due to associated environmental pollution and health risks. Among the toxins produced by these blooms, microcystins, particularly Microcystin MC-LR, pose significant threats to ecosystems and human health. This study investigates the efficiency of degrading microcystins, particularly Microcystin MC-LR, using two catalytic methods: whole-cell and pure enzyme catalysis, both relying on the mlrA gene. The research successfully engineered an Escherichia coli strain with the capability to degrade MC-LR, achieving remarkable results with the whole-cell catalysis approach. Within a 24-hour period, the concentration of MC-LR dropped from 5 μg/mL to 0.681 μg/mL, demonstrating an efficiency of 84%. In contrast, enzymatic degradation yielded less satisfactory outcomes, degrading only 100 ng/mL within the same timeframe. The whole-cell catalysis approach exhibits clear advantages, including cost-effectiveness, making it a promising option for industrial applications. This technology holds potential for safeguarding water resources and public health by addressing algal toxin contamination.

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Impacts of algal organic matter and humic substances on microcystin-LR removal and their biotransformation during the biodegradation process

The application of bioaugmentation (i.e., injection of contaminant-degrading microorganisms) has shown its potential to remove harmful cyanotoxins like microcystin-LR (MC-LR) from drinking water sources. However, the natural organic matter (NOM) present in both natural and engineered water systems might affect the bacterial biodegradation of MC-LR. Therefore, for the successful application of bioaugmentation for MC-LR removal in water treatment, it is important to understand NOM effects on MC-LR biodegradation. In this study, the impact of NOM [algal organic matter (AOM) and humic substances (HS)] on MC-LR biodegradation was evaluated in the presence of varying concentrations of NOM by monitoring MC-LR biodegradation kinetics. The changes in NOM composition during MC-LR biodegradation were also characterized by a five-component Parallel factor (PARAFAC) model using 336 excitation-emission matrix (EEM) spectra collected at different sampling points. Our results showed decreases in MC-LR biodegradation rate of 1.6-and 3.4-fold in the presence of AOM and HS, respectively. The expression of the functional mlrA gene exhibited a similar trend to the MC-LR degradation rate at different NOM concentrations. EEM-PARAFAC analyses and NOM molecular size fractionation results indicated a relatively greater production of terrestrial humic-like components (57%) and a decrease of protein-like components. Two-dimensional correlation spectroscopy (2D-COS) analyses further confirmed that low molecular weight protein-like components were initially utilized by bacteria, followed by the formation of higher molecular weight humic-like components, likely due to microbial metabolism.

Biodegradation of Nodularin by a Microcystin-Degrading Bacterium: Performance, Degradation Pathway, and Potential Application.

Currently, studies worldwide have comprehensively recognized the importance of Sphingomonadaceae bacteria and the mlrCABD gene cluster in microcystin (MC) degradation. However, knowledge about their degradation of nodularin (NOD) is still unclear. In this study, the degradation mechanism of NOD by Sphingopyxis sp. m6, an efficient MC degrader isolated from Lake Taihu, was investigated in several aspects, including degradation ability, degradation products, and potential application. The strain degraded NOD of 0.50 mg/L with a zero-order rate constant of 0.1656 mg/L/d and a half-life of 36 h. The average degradation rate of NOD was significantly influenced by the temperature, pH, and initial toxin concentrations. Moreover, four different biodegradation products, linear NOD, tetrapeptide H-Glu-Mdhb-MeAsp-Arg-OH, tripeptide H-Mdhb-MeAsp-Arg-OH, and dipeptide H-MeAsp-Arg-OH, were identified, of which the latter two are the first reported. Furthermore, the four mlr genes were upregulated during NOD degradation. The microcystinase MlrA encoded by the mlrA gene hydrolyzes the Arg-Adda bond to generate linear NOD as the first step of NOD biodegradation. Notably, recombinant MlrA showed higher degradation activity and stronger environmental adaptability than the wild strain, suggesting future applications in NOD pollution remediation. This research proposes a relatively complete NOD microbial degradation pathway, which lays a foundation for exploring the mechanisms of NOD degradation by MC-degrading bacteria.

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.

Biofilm formation by Non-O157 Shiga toxin-producing Escherichia coli in monocultures and co-cultures with meat processing surface bacteria

This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by non-O157 Shiga toxin-producing Escherichia coli (STEC), and potential links between biofilm formation by STEC and biofilm-related genes in their genomes. Biofilm development by 50 MPB and 6 STEC strains in mono- and co-cultures was assessed by the crystal violet staining method, and their expression of curli and cellulose was determined using the Congo red agar method. Genes (n = 141) associated with biofilm formation in the STEC strains were profiled. Biofilm formation in general correlated with cellulose and curli expression in both mono- and co-cultures. Most MPB strains had antagonistic effects on the biofilm formation of the STEC strains. Of the genes investigated, 81% were common among the STEC strains and there seems to be a gene-redundancy in biofilm formation. The inability of the O26 strain to form biofilms could be due to mutations in the rpoS gene. Truncation in the mlrA gene in the O145 strain seems not affecting its biofilm formation alone or with MPB. The O45 strain, despite having the greatest number of biofilm-related genes, did not form measurable biofilms. Overall, biofilm formation of STEC was affected by curli-cellulose expression and companion strains.

Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

Simultaneous Removal of the Freshwater Bloom-Forming Cyanobacterium Microcystis and Cyanotoxin Microcystins via Combined Use of Algicidal Bacterial Filtrate and the Microcystin-Degrading Enzymatic Agent, MlrA.

Freshwater cyanobacterial blooms (e.g., Microcystis blooms) constitute a major global environmental problem because of their risks to public health and aquatic ecological systems. Current physicochemical treatments of toxic cyanobacteria cause the significant release of cyanotoxin microcystins from damaged cells. Biological control is a promising eco-friendly technology to manage harmful cyanobacteria and cyanotoxins. Here, we demonstrated an efficient biological control strategy at the laboratory scale to simultaneously remove Microcystis and microcystins via the combined use of the algicidal bacterial filtrate and the microcystin-degrading enzymatic agent. The algicidal indigenous bacterium Paenibacillus sp. SJ-73 was isolated from the sediment of northern Lake Taihu, China, and the microcystin-degrading enzymatic agent (MlrA) was prepared via the heterologous expression of the mlrA gene in the indigenous microcystin-degrading bacterium Sphingopyxis sp. HW isolated from Lake Taihu. The single use of a fermentation filtrate (5%, v/v) of Paenibacillus sp. SJ-73 for seven days removed the unicellular Microcystis aeruginosa PCC 7806 and the native colonial Microcystis strain TH1701 in Lake Taihu by 84% and 92%, respectively, whereas the single use of MlrA removed 85% of microcystins. Used in combination, the fermentation filtrate and MlrA removed Microcystis TH1701 and microcystins by 92% and 79%, respectively. The present biological control thus provides an important technical basis for the further development of safe, efficient, and effective measures to manage Microcystis blooms and microcystins in natural waterbodies.

Optimization of microcystin biodegradation by bacterial community YFMCD4 using response surface method

The increasing production of microcystin-LR (MC-LR) causing animal and human health issues is found in eutrophic water bodies, marine habitats and desert environments. The health threat posed by MC-LR has led to the establishment of World Health Organization’s water guideline value of 1 μg/mL. Combating this has increased the search for cost-effective approach to degrade MC-LR. The study aimed to optimize the MC-degrading environmental factors of bacterial community YFMCD4. Response surface methodology (RSM) was employed to evaluate the influence of varying temperatures, pH and initial MC-LR concentration on the biodegradation efficiency of MC-LR by bacterial community YFMCD4. The optimal MC-LR biodegradation environmental factors were found to be 30 °C, pH 7 and 2 μg/mL initial MC-LR. The biodegradation rate reached 100% after 10 h. YFMCD4 mainly consisted of genera Alacligenes, Sphingobacterium and Pseudomonas using High-throughput pyrosequencing technology. The mlrA gene encoding MlrA enzyme considered most important for MC-LR biodegradation was obtained from YFMCD4. Data demonstrated that the bacterial structure and biodegradation efficiency of YFMCD4 varied with the change of environmental factors including temperature, pH and MC-LR concentrations. RSM is considered a good method to examine the optimal biodegradation environmental conditions for MC-LR. To date, RSM and High-throughput pyrosequencing technology are employed to optimize the biodegradation conditions (30 °C, pH 7 and 2 μg/mL initial MC-LR) and analyze the structure of bacterial community for the first time.

How Copepods Can Eat Toxins Without Getting Sick: Gut Bacteria Help Zooplankton to Feed in Cyanobacteria Blooms.

Toxin-producing cyanobacteria can be harmful to aquatic biota, although some grazers utilize them with often beneficial effects on their growth and reproduction. It is commonly assumed that gut microbiota facilitates host adaptation to the diet; however, the evidence for adaptation mechanisms is scarce. Here, we investigated the abundance of mlrA genes in the gut of the Baltic copepods Acartia bifilosa and Eurytemora affinis during cyanobacteria bloom season (August) and outside it (February). The mlrA genes are unique to microcystin and nodularin degraders, thus indicating the capacity to break down these toxins by the microbiota. The mlrA genes were expressed in the copepod gut year-round, being >10-fold higher in the summer than in the winter populations. Moreover, they were significantly more abundant in Eurytemora than Acartia. To understand the ecological implications of this variability, we conducted feeding experiments using summer- and winter-collected copepods to examine if/how the mlrA abundance in the microbiota affect: (1) uptake of toxic Nodularia spumigena, (2) uptake of a non-toxic algal food offered in mixtures with N. spumigena, and (3) concomitant growth potential in the copepods. The findings provide empirical evidence that the occurrence of mlrA genes in the copepod microbiome facilitates nutrient uptake and growth when feeding on phytoplankton mixtures containing nodularin-producing cyanobacteria; thus, providing an adaptation mechanism to the cyanobacteria blooms.

Taxonomic and Genotypical Heterogeneity of Microcystin degrading Bacterioplankton in Western Lake Erie

Microcystins (MCs) are among the predominant cyanotoxins that are primarily degraded by heterotrophic bacteria in various freshwater environments, including Lake Erie, a Laurentian Great Lake. However, despite the prevalence of MCs in Lake Erie basins, our knowledge about the taxonomic diversity of local MC-degrading bacteria is largely limited. The current study obtained thirty-four MC-degrading bacterial pure isolates from Lake Erie surface water and characterized their taxonomical and phenotypic identities as well as their MC-degradation rates under different pH, temperature, availability of organic substrates and with other MC-degrading isolates. Obtained MC-degrading isolates included both Gram-positive (18 isolates of Actinobacteria and Firmicutes) and Gram-negative bacteria (16 isolates of Gamma-proteobacteria); and 7 of these isolates were motile, and 13 had the capacity to form biofilms. In general, MC-degradation rates of the isolates were impacted by temperature and pH but insensitive to the presence of cyanobacterial exudates. At the optimal temperature (30–35°C) and pH (7–8), individual isolates degraded MC-LR, the most abundant MC isomer, at an average of 0.20 µg/mL/hr. With additions of cyanobacterial exudates, only Pseudomonas sp. LEw-2029, a non-motile biofilm maker, showed increased MC degradation (0.25 µg/mL/hr). Five out of nine tested dual culture mixtures showed rises in MC degradation rates than their corresponding monocultures; the highest rate reached 0.40 µg/mL/hr for the pair LEw-(1132 + 2029). PCR amplification of mlrA genes yielded negative results for all isolates; subsequent enzyme assay-Mass Spectrum analysis identified no product associated with the mlr gene-based MC degradation pathway. Collectively, our results demonstrated that a diversity of indigenous Lake Erie bacteria can degrade MCs via a novel mlr-independent pathway. Obtained MC degraders, especially Pseudomonas sp. LEw-2029, may serve as candidates for the development of biological filters to remove cyanotoxins in water treatment systems.

Impact of algal organic matter on the performance, cyanotoxin removal, and biofilms of biologically-active filtration systems

The occurrence of harmful algal blooms dominated by toxic cyanobacteria has induced continuous loadings of algal organic matter (AOM) and toxins in drinking water treatment plants. However, the impact of AOM on the active biofilms and microbial community structures of biologically-active filtration (BAF), which directly affects the contaminant removal, is not well understood. In this study, we systematically examined the effects of AOM on BAF performance and bacterial biofilm formation over 240 days, tracing the removal of specific AOM components, a cyanotoxin [microcystin-LR (MC-LR)], and microbial community responses. The component analysis (excitation and emission matrix analysis) results for AOM revealed that terrestrial humic-like substances showed the highest removal among all the identified components and were strongly correlated to MC-LR removal. In addition, reduced empty bed contact time and deactivation of biofilms significantly decreased BAF performances for both AOM and MC-LR. The active biofilm, bacterial community structure, and mlrA gene (involved in microcystin degradation) abundance demonstrated that bacterial biofilm composition responded to AOM and MC-LR, in which Rhodocyclaceae, Saprospiraceae, and Comamonadaceae were dominant. In addition, MC-LR biodegradation appeared to be more active at the top than at the bottom layer in BAF. Overall, this study provides deeper insights into the role of biofilms and filter operation on the fate of AOM and MC-LR in BAF.

Isolation of a Novel Microcystin-Degrading Bacterium and the Evolutionary Origin of mlr Gene Cluster.

The mlr-dependent biodegradation plays an essential role in the natural attenuation of microcystins (MCs) in eutrophic freshwater ecosystems. However, their evolutionary origin is still unclear due to the lack of mlr gene cluster sequences. In this study, a Sphingopyxis sp. strain X20 with high MC-degrading ability was isolated, and the mlrA gene activity was verified by heterologous expression. The whole sequence of the mlr gene cluster in strain X20 was obtained through PCR and thermal asymmetric interlaced (TAIL)-PCR, and then used for evolutionary origin analyses together with the sequences available in GenBank. Phylogenetic analyses of mlr gene clusters suggested that the four mlr genes had the same origin and evolutionary history. Genomic island analyses showed that there is a genomic island on the genome of sphingomonads that is capable of degrading MCs, on which the mlr gene cluster anchors. The concentrated distribution of the mlr gene cluster in sphingomonads implied that these genes have likely been present in the sphingomonads gene pool for a considerable time. Therefore, the mlr gene cluster may have initially entered into the genome of sphingomonads together with the genomic island by a horizontal gene transfer event, and then become inherited by some sphingomonads. The species other than sphingomonads have likely acquired mlr genes from sphingomonads by recently horizontal gene transfer due to the sporadic distribution of MC-degrading species and the mlr genes in them. Our results shed new light on the evolutionary origin of the mlr cluster and thus facilitate the interpretation of characteristic distribution of the mlr gene in bacteria and the understanding of whole mlr pathway.

Enhanced removal of Microcystis bloom and microcystin-LR using microcosm constructed wetlands with bioaugmentation of degrading bacteria

The prevalence of cyanobacterial bloom (Cyano-bloom) and hepatotoxic microcystin (MC) pollution caused by eutrophication poses serious problems to aquatic ecosystems and public health. However, conventional water treatment technologies are inefficient for removing cyanotoxins. In this study, the performance of microcosm constructed wetlands (CWs) in the removal of Cyano-bloom, microcystin-LR (MC-LR), and nutrients was investigated following repeated loading of pollutants. The effects of plant and bioaugmentation of selected MC-LR degrading bacteria on removal efficiency, degrading gene mlrA abundance, and bacterial community structure were examined. More than 90% of the MC-LR and chlorophyll-a was eliminated by CWs after 3 d of hydraulic retention time (HRT) without a lag phase. No significant differences between planted and unplanted CWs were found in the MC-LR and Cyano-bloom removal and mlrA gene abundance. Nevertheless, the plants improved nutrient removal to reduce eutrophication. Bioaugmentation markedly enhanced the degradation of MC-LR from 16.7 μg L−1 to below the threshold value within 12 h, which could help shorten the HRT of CWs by increasing functional MC-LR degrading bacteria. In the soil of CWs, the following six bacterial genera with MC-LR-degrading potential were found: Sphingopyxis, Methylotenera, Pseudomonas, Methylosinus, Novosphingobium, and Sphingomonas. Among them, the first three also significantly proliferated in CWs with bioaugmentation during MC-LR degradation, indicating their high adaptability and MC-LR removal contribution. These results suggested that CWs could provide suitable conditions for MC-LR degrading microorganism proliferation, and CWs with bioaugmentation could be effective and practical measures for the remediation of eutrophication and MC pollution.

Sulfamethoxazole - Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate.

The high frequency of prophage insertions in the mlrA gene of clinical serotype O157:H7 isolates renders such strains deficient in csgD-dependent biofilm formation but prophage induction may restore certain mlrA properties. In this study we used transcriptomics to study the effect of high and low sulfamethoxazole–trimethoprim (SMX-TM) concentrations on prophage induction, biofilm regulation, and virulence gene expression in strain PA20 under environmental conditions following 5-hour and 12-hour exposures in broth or on agar. SMX-TM at a sub-lethal concentration induced strong RecA expression resulting in concentration- and time-dependent major transcriptional shifts with emphasis on up-regulation of genes within horizontally-transferred chromosomal regions (HTR). Neither high or low levels of SMX-TM stimulated csgD expression at either time point, but both levels resulted in slight repression. Full expression of Ler-dependent genes paralleled expression of group 1 pch homologues in the presence of high glrA. Finally, stx2 expression, which is strongly dependent on prophage induction, was enhanced at 12 hours but repressed at five hours, in spite of early SOS initiation by the high SMX-TM concentration. Our findings indicate that, similar to host conditions, exposure to environmental conditions increased the expression of virulence genes in a clinical isolate but genes involved in the protective biofilm response were repressed.

Elucidating the Regulatory Functions of MlrA Originated from Novosphingobium sp. THN1 in Microcystin-LR Degradation

Microcystin-LR (MC-LR), produced by harmful cyanobacteria, seriously endangers animals and humans. Biodegradation appears as the major pathway for natural MC-LR attenuation. To elucidate the regulatory function of mlrA gene of Novosphingobium sp. THN1 (i.e., THN1-mlrA gene) in MC-LR biodegradation, this study constructed a recombinant bacterium and succeeded in heterlogously expressing the mlrA of THN1 strain (i.e., THN1-MlrA enzyme). The recombinant mlrA exhibited the activity for smoothly degrading 20 μg mL-1 of MC-LR at an average rate of 0.16 μg mL-1 h-1 within 80 h. Mass spectrum analysis confirmed that recombinant mlrA hydrolyzed cyclic MC-LR by cleaving the peptide bond between Adda and arginine residue and generated linearized MC-LR as primary intermediate. Such linearization for MC-LR catalyzed by THN1-MlrA enzyme was particularly important during MC-LR biodegradation process, because it opened the highly-stable cyclic structure of MC-LR and caused substantial detoxification. These findings for the first time manifested that mlrA gene homolog of Novosphingobium genus conserved its original catalytic function as described elsewhere. This study expanded the knowledge on the function of mlrA homologs from various natural habitats, and facilitated the understanding on the fate and biological attenuation mechanisms of MC-LR in Lake Taihu, China, where THN1 strain is indigenous.

Diversity and temporal shifts of the bacterial community associated with a toxic cyanobacterial bloom: An interplay between microcystin producers and degraders

The biodegradation of microcystins (MCs) by bacteria constitutes an important process in freshwater ecosystems to prevent the accumulation of toxins. However, little is known about the diversity and the seasonal dynamics of the bacterial community composition (BCC) involved in the degradation of MCs in nature. To explore these BCC shifts, high-throughput sequencing was used to analyse the 16S rRNA, mcyE and mlrA genes during a year in a freshwater reservoir with a toxic cyanobacterial bloom episode. The analysis of the mcyE and mlrA genes from water samples revealed the coexistence of different MC-producing and MC-degrading genotypes, respectively. The patchy temporal distribution of the mlrA genotypes (from the families Sphingomonadaceae and Xanthomonadaceae) suggests their dissimilar response to environmental conditions and the influence of other factors besides the MCs that may control their presence and relative abundance. During the maximum toxic cyanobacterial biomass and cell lysis, other bacterial taxa that lack mlr genes increased their relative abundance. Among these bacteria, those with a recognized role in the degradation of xenobiotic and other complex organic compounds (e.g., orders Myxococcales, Ellin6067, Spirobacillales and Cytophagales) were the most representative and suggest their possible involvement in the removal of MCs in the environment.

Heterologous expression of mlrA gene originated from Novosphingobium sp. THN1 to degrade microcystin-RR and identify the first step involved in degradation pathway

Information on the catalytic role of mlrA gene-encoded enzyme (MlrA) in microcystin-RR (MC-RR) biodegradation was limited. This study succeeded in expressing mlrA homolog of Novosphingobium sp. THN1 in heterologous host for the first time, by constructing a recombinant bacterium. Mass spectrometric analysis showed that the recombinant MlrA hydrolyzed MC-RR into linear intermediate product by cleaving the peptide bond between Adda and arginine residue, greatly detoxifying MC-RR. This finding clearly manifested that the MlrA homolog of THN1 strain possesses its original catalytic function, and ring-opening constituted the first step in MC-RR biodegradation pathway of THN1 strain. Moreover, MC-RR degradation by intact recombinant cells and cell-free crude enzyme (CE) from recombinant was compared. Results exhibited that intact recombinant was able to degrade 20 μg mL−1 MC-RR more quickly than CE, with the maximum rate of 9.22 μg mL−1 h−1 in the first 8 h. Thus, this study provided new insights on the catalytic activity and roles of MlrA originated from THN1 strain in MC-RR biodegradation process, which lay a foundation for efficiently removing and detoxifying MC-RR, and exploring downstream steps in MC-RR biodegradation pathway of THN1 strain.

Microcystin-LR Biodegradation by Bacillus sp.: Reaction Rates and Possible Genes Involved in the Degradation

Harmful cyanobacteria blooms may deteriorate freshwater environments, leading to bad water quality that can adversely affect the health of humans, animals, and aquatic life. Many cyanobacteria can produce toxic metabolites, with Microcystin-LR (MC-LR) being the most commonly detected cyanotoxin in fresh water bodies. In this study, a MC-LR degrading Bacillus sp. strain was isolated from Hulupi Lake (HLPL), Taiwan and tested for its degradability of the cyanotoxin. The results showed that the degradation of Microcystin-LR by the isolated Bacillus sp. was temperature-dependent with an optimum MC-LR removal at 37 °C and a first order degradation constant rate for 0.22 day−1. The degradation rate was also found to increase with decreasing MC-LR concentrations and increasing Bacillus sp. concentrations. Biomolecular monitoring of three types of genes (mlrA, CAAX, and GST) involved in the degradation indicated that mlrA, and CAAX genes were present in the indigenous bacteria in HLPL water samples. However, for the isolated Bacillus sp. strain, only CAAX genes were detected. The absence of the mlrA gene in the isolated Bacillus sp. strain shows that the degradation of MC-LR does not necessarily follow the pathways with mlrA, and can also follow the pathways involved with CAAX type II amino-terminal protease.