Elucidation of Microcystin-LR Degrading Mechanism of Bacillus cereus

Abstract

Microcystin-LR (MC-LR) is a cyclic peptide produced as a secondary metabolite of certain freshwater cyanobacteria species such as, Microcystis, Anabaena, and Oscillatoria. Biodegradation of MC-LR by heterotrophic bacteria have been accepted as a reliable and cost effective method to treat MC-LR contamination. To date, more than 30 different bacterial genera have been recorded as potential MCs degraders. However, limited information are available on MC-LR degradation mechanism of bacteria. Intracellular MC-LR degradation by enzymes encoded by mlr A,B,C and D genes is the only hypothesis accepted so far. Hence, the present study focused on the elucidation of MC-LR degradation mechanism of KJ954304 Bacillus cereus 12GK strain which was previously isolated and characterized by authors as an efficient MC-LR degrader. Overnight grown and starved B. cereus bacterial suspension (0.5 µl) was inoculated into 100 ml of filter sterile (0.2 μm) Beira lake water containing MC-LR at a final concentration of 5 µgml -1 . Control sample was prepared without bacterial inoculation. All flasks were incubated at 28°C and shaken at 100 rpm for 3 days. Following three days of incubation 0.5 ml sub sample aliquot was removed from both experimental and control flasks and frozen at (-20°C). Then 45 ml of experimental sample was kept aside as initial experimental sample and remaining 45 ml of sample was filtered under sterile conditions using 0.2 μm filter to remove bacterial cells. Then the filtrate was placed immediately in ice to prevent denature of enzymes. Subsequently, MC-LR was spiked to both initial experimental samples and filtered samples at a final concentration of 5µgml-1, and incubated at 28 0 C, 100 rpm for 3 days. 1ml aliquots were removed from initial experimental sample and filtrate sample for 0-3 days of incubation. These samples were frozen and processed for High Performance Liquid Chromatography (HPLC) analysis. A PCR analysis was carried out to detect the presence of MC-LR degrading mlr A,B,C and D genes in B. cereus. Amplifications were performed in 50 µL volumes, containing 1 mM of each primer. A GeneAmps 2400 PCR System was utilized for the amplifications.At the end of 3 days of experiment MC-LR concentration of the initial experimental sample with bacteria was 1.8 µgml -1 whereas the filtrate sample without bacteria had 4.8 µgml -1 of MC-LR. Furthermore, the PCR study confirmed the presence of mlr A,B,C and D genes in B. cereus. Thus, MC-LR degradation is performed as an intracellular degradation by B. cereus with the involvement of intracellular enzymes.

A novel cyanolytic bacterium, Pseudomonas fluorescens BG‐E as a potential biological control agent for freshwater bloom‐forming cyanobacteria Pseudanabaena spp.

“…The bacterial strain BG‐E with the potential to lyse cyanobacteria was screened for the presence of the microcystin‐degrading gene cluster ( mlr ABCD) by using PCR. Genomic DNA was amplified with oligonucleotide primers for mlr A, mlr B, mlr C, and mlr D genes (Idroos & Manage, 2018) using FastGene® Taq ReadyMix (Nippon Genetics) following instructions from the manufacturer with an initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 5 °C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 2 min.…”

“…Additionally, some of these cyanolytic bacteria have the potential to degrade cyanotoxins such as MCs and suppress blooms instantaneously (Li et al, 2016). Consequently, considerable attention has been paid to heterotrophic bacteria in freshwater due to their potential as agents to control or terminate the excessive growth of cyanobacteria (Van Le et al, 2022; Wang et al, 2020; Zhang et al, 2019) and to degrade cyanotoxins (Chaffin et al, 2022; Idroos & Manage, 2018; Nájera et al, 2017). Although the identification of an effective biological control agent is challenging, recognizing and isolating bacteria with the ability to lyse Microcystis sp.…”

Biodegradation of the cyanotoxin cylindrospermopsin by Bacillus cereus, Micrococcus luteus and Alcaligenes faecalis 

“…Four bacterial strains-two strains of Bacillus cereus named B. cereus-S (BC-S) and B. cereus-Y (BC-Y), M. luteus (ML-M), and A. faecalis (AF-M)-which have been isolated from previous studies (Liyanage & Manage, 2016;Idroos & Manage, 2018;Ekanayake & Manage, 2020) were tested to evaluate their ability to degrade CYN, following the method described by Manage et al, (2016) and Manage et al, (2009). A loop-full of each bacterial isolate was inoculated in 5 mL of sterile Lurial Broth (LB) and incubated overnight at 28 0 C and at pH 7.…”

“…Triplicate samples were prepared for each bacterial strain and incubated at 28°C for 14 days. Sample aliquots (1000 µL) were removed at intervals of two days for 14 days, and the samples were frozen immediately at -20 0 C. For the analysis of CYN, the samples were freeze-dried, reconstituted in 1 mL of 80% (v/v) HPLC grade methanol followed by centrifugation at 6000 rpm for 20 min and the supernatant was removed, filtered through a 0.2 μm nylon syringe filter and 50 μL of the sample were subjected to PDA-HPLC analysis (Idroos & Manage, 2018).…”

“…The objective of the present study is to determine the efficacy of degrading CYN by four bacterial strains namely, Bacillus cereus-S (BC-S), Bacillus cereus-Y (BC-Y), Micrococcus luteus (ML-M), and Alcaligenes faecalis (AF-M) which were previously proven to degrade several other environmental pollutants including pesticides (Geed et al, 2017), dyes (Thakur et al, 2014;Ekanayake & Manage, 2017), polycyclic aromatic hydrocarbons (Liyanage & Manage, 2016;Dharmadasa et al, 2017;Idroos & Manage, 2017) and antibiotics (Idroos & Manage, 2014). The BC-S strain employed in this study was isolated from the surface water of the Giradurukotte reservoir in Sri Lanka during a previous study and has been proven to degrade MC-LR completely within 8 days (Idroos & Manage, 2018). The other Bacillus strain used in this study, BC-Y, was previously isolated from crude oil contaminated surface water and identified as a hydrocarbon degrader (Liyanage & Manage, 2016).…”