Genomic insight for Algicidal activity in Rhizobium sp. (AQ_MP)


 Occurrence of Harmful Algal Blooms (HABs) creates a threat to aquatic ecosystem affecting the existing flora and fauna. Hence, the mitigation of HABs through an eco-friendly approach remains a challenge for environmentalists. The present study provides the genomic insights of Rhizobium sp. (AQ_MP), an environmental isolate that showed the capability of degrading Microcystis aeruginosa (Cyanobacteria) at laboratory scale. Genome sequence analysis of Rhizobium sp. (AQ_MP) was performed to determine the algal lysis properties and toxin degradative pathway. It is envisaged that Rhizobium sp. (AQ_MP) secreted CAZymes like Glycosyltransferases (GT), Glycoside Hydrolases (GH), polysaccharide lyases (PL), which allowed algal polysaccharide degradation (lysis) and enabled nutrient release for the subsequent growth of Rhizobium sp. (AQ_MP) Genome analysis also showed the presence of the glutathione metabolic pathway, which is the biological detoxification pathway responsible for microcystin degradation. The conserved region mlrC, a microcystin toxin degrading responsible gene, was also annotated in Rhizobium sp. (AQ_MP). This study confirmed that Rhizobium sp. (AQ_MP) harbours a wide range of crucial enzymes released for lysis of Microcystis aeruginosa (M. aeruginosa) cells and also for degradation of microcystin toxin. This study thus find promiscuity for scaling the lab based analysis to field level in future.


Introduction
Cyanobacteria are the photosynthetic organisms found in both freshwater and marine environment (Pal, M et al., 2018), which can be pelagic and benthic (Wehr, J.D et al., 2015). Although cyanobacteria have few positive trends such as xing atmospheric nitrogen but on the other hand, few species release toxic secondary metabolites such as dermatoxins, hepatotoxins, cytotoxins, and neurotoxins (Carmichael, W.W., 2001, Pearson, L., et al., 2010, Schmidt, J.R., et al., 2014. Cyanobacteria have another ability to avoid predation by grazers (Lampert, W., 1987). They can form elongated shapes, colonies and release some toxic secondary metabolites. The increased anthropogenic activities these days have increased the concentration of nitrogen and phosphate in water bodies which in the major contributor to the proliferation of algal bloom (J. R., et al., 2014). Algal bloom affects the entire water body by hindering light penetration and toxin release (Paerl, H.W., et al., 2011, Pal M., et al., 2020. The recreational activities and drinking of these water affected by algal blooms often disturb animal health (Pal M., et al., 2020).
Some of these algal bloom species release lethal toxins. The Microcystis species, such as M. aeruginosa, M. fosaquae, M. wesenbergii, M. ichthyoblabe, and M. phertaare are the leading cause of almost 90% of the harmful algal blooms in freshwater. M. aeruginosa is the most commonly observed cyanobacterial species causing harmful algal blooms and releases toxin, mainly microcystins (MCs) (Kim, M., et al., 2019), which are neurotoxin and hepatotoxins. Till date, more than 90 types of microcystins are released. Still, microcystin-LR is the most abundant and highly toxic variant (Pal M., et al., 2020). Toxic mechanism of MCs is due to the disruption of cytoskeleton formation and inhibition of protein phosphatase. In humans, MCs can enter through toxin-contaminated water or diet; they can cause oxidative stress, leading to cell damage (J. R., et al., 2014). Microcystins are readily water-soluble because their log of octanol and water distribution ration is approximately -1 (pH 7). The essential binding sites of microcystins to the protein phosphatase are methyl-dehydroalanine (Mdha) and 3-amino-9-methyoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (ADDA) groups. Microcystin toxicity depends on the amino acid combination present at the two different positions of the peptide ring. Microcystin-LR has amino acid leucine and arginine at the variable peptide ring, and Microcystin-RR has arginine and arginine (Rinehart, K.L., et al., 1994, Corbel, S., et al., 2014. Zhang, J., et al., 2017 reported an innate effective bacterium of Sphingopyxis sp. having microcystin degrading capability. It contained enzymes of microcystin degradation i.e. mlrA, mlrC and mlrD. Mann, A.J., et al., 2013 reported bacterium Formosa agariphila (KMM 3901T) has a broad potential of algal polysaccharide degradation. Similarly, in this study, we have isolated a bacteria which has algicidal and microcystin degradation property. We observed the mechanism of interaction via; SEM analysis and genome sequencing. Genome sequence data analysis con rms the presence of pathways and genes responsible for algal lysis and conserved region for microcystin degradation.

Isolation and cultivation of M. aeruginosa and Rhizobium (AQ_MP) used for interaction studies
Microcystis aeruginosa was isolated from water samples of Ambazari Lake, Nagpur, Maharashtra. Culture was maintained by subculturing for 30-35 days as de ned by (Sangolkar L. N, et al., 2009). M. aeruginosa was poured rst with the OD 678 0.03, cultured in 250 mL of conical asks using BG-11 medium under continuous cool uorescent light (12:12 light and dark cycle, 3000 lux) at 25±1°C and mechanically shaken a day thrice. Different bacterial cultures were isolated, puri ed and characterized from the lake water sample published in our previous studies (Pal, M., et al., 2018). Fresh bacterial culture was prepared by inoculation of Rhizobium sp. (AQ_MP) into 250 ml conical ask containing 100ml of Luria broth media and kept overnight in a shaking incubator (120 rpm, 30 C) for 24 hours. Growth curve of Rhizobium sp. (AQ_MP) also analysed (Supplementary Figure 1).
2.2 aeruginosa cells after exposure to Rhizobium sp. (AQ_MP) : Lysis of algae M. aeruginosa (100 ml) suspension were interacted with 10 ml of 1 OD 600 bacterial suspension (Rhizobium). The experiment was done for ten days, where control and treated samples were collected every day. Samples (1ml) were centrifuged at 10000 rpm for 10 mins and xed in 5% glutaraldehyde, and kept at 4 C for Scanning Electron Microscopy (SEM) analysis. Cells (control and experimental) were centrifuged at 10000 rpm and suspended and xed with 5% v/v glutaraldehyde in 0.1 M phosphate buffer (30 min). Fixed control and experimental cells were centrifuged again, and the supernatant was discarded. Cell pellets were washed three times with 0.1M phosphate buffer (15 min); dehydration was done with 35% ethanol (15 min), 50% ethanol (15 min), 75% ethanol (15 min), 95% ethanol (15 min), and two times with 100% ethanol (15 min). The gold coating of samples was done using Tescan SEM equipment. The cells were then examined in a Vegag3 software, operating at 5.0 kV by a Germany scanning electron microscope (Kim, M.,et al., 2020). The total chlorophyll concentration was estimated for the algae-bacteria mixture for ten days by a method described by (Gupta, S. and Pawar, S.B., 2018), wherein ltered dried biomass was extracted with 80% of ice-chilled acetone for estimation of chlorophyll using wavelength 663.2nm and 646.8nm.

Whole-genome sequencing and annotation of Rhizobium (AQ_MP):
The qualities and quantities of the received bacterial DNA samples were checked by resolved on1% Agarose gel followed by quanti cation using NanoDrop. The gDNA samples were identi ed on the basis of molecular identi cation by targeting the bacterial 16S gene using Sanger sequencing technique. PCR ampli ed the fragment of the bacterial 16S gene. A single distinct amplicon band of PCR was observed in the agarose gel. Speci c primer was used for DNA sequencing reaction of PCR amplicons. QC passed genomic DNA sample was used for the paired-end sequencing library preparation (after con rmation), using Illumina TruSeqNano DNA Library Prep Kit (Srinivasan, V.B. andRajamohan, G., 2020, Tikariha, H., et al., 2016). Gene prediction and functional annotation were made using Rapid Annotation using Subsystem Technology (RAST) server (Overbeek, R., et al., 2014). CG Viewer server was also used to create a circular genome that allowed to visualize sequence feature information in the context of sequence analysis outcomes. In this server only, PROKKA annotation was also done to merge CDS, tRNA, tmRNA and rRNA subunits. InterProscan database using pfam system (Patel, D, D et al., 2014) was used to check the presence of the genetic domain of microcystin protein family. Dbcan database was used to check the annotate CAZymes.

Interaction study of Microcystis aeruginosa and Rhizobium (AQ_MP):
In this study, control and experimental Microcystis aeruginosa culture with Rhizobium sp. (AQ_MP) culture was kept for 10 days to check the interaction and degradation of M. aeruginosa cells. After the collection of samples, SEM analysis showed the contact between M. aeruginosa and Rhizobium sp. (AQ_MP) cells. In ten days of interaction, M. aeruginosa cells were lysed in ask experiments, as demonstrated in our previous studies (Pal M., et al. 2018). Many studies have already been suggested that M. aeruginosa have large mucilaginous aggregates comprised of a mucus substance called phycosphere. This mucous region typically comprises associated epiphytic bacteria (Kim, M.,et al., 2019). (Zhang, H., et al., 2011, Gumbo, J.R. and Cloete, T.E., 2011) already published some data on the interaction mechanism between bacteria and M. aeruginosa that depicts the M. aeruginosa cell membrane's damage, followed by the release of some extracellular substances. These extracellular substances are useful nutrients for bacteria growth. This bacteria-cyanobacteria sometimes shows epiphytic relationships, where the dominant species ourish the most. Total Chlorophyll estimation was also estimated which showed that the chlorophyll level was decreased at the end of the 10 th day ( Figure   1).
3.2 Lysis of aeruginosa cells exposed to Rhizobium sp. (AQ_MP) and it's mechanism of action: SEM was performed to analyze cellular interactions between the Rhizobium sp. (AQ_MP) and M. aeruginosa cells. On the rst day of experiment, M. aeruginosa was observed as a dominant organism. As the incubation continues, the Rhizobium sp. (AQ_MP) cells immersed as in dominant organism indicating towards the lysis of M. aeruginosa. Figure  Our previous study showed that Rhizobium sp. (AQ_MP) cells used microcystin toxin as a carbon source (Pal M., et al., 2018). The present study is an advancement to the previous ndings where cell lysis occurred due to enhanced bacterial population. Increased Rhizobium sp. (AQ_MP) cells cause light hindrance, which affects the M. aeruginosa cell growth. Rhizobium sp. (AQ_MP) cells also release lytic enzymes, which leads to the destruction of the M. aeruginosa cell wall. This destructed M. aeruginosa cell becomes a nutrient for the growth of Rhizobium sp. (AQ_MP) cells and continues the deterioration of M. aeruginosa cell.

Whole-genome sequencing statistics:
The ltered high-quality PE reads of the bacterial samples mentioned were assembled into scaffolds using SPAdes assembler (v-3.13.0). Nanodrop reading was observed as 152 ng/µl. Total data was 616 Mb; the total number of bases were 615,855,661, total number of reads were observed 2,069,397, total number of scaffolds was 122. The average scaffold size was (bp) 43,485. Max and min scaffold size was 620,217bp and 200bp. Whole-genome was submitted to NCBI/Genbank under the accession number JACJVI010000000 as Rhizobium sp. (AQ_MP). After getting the NCBI genome data, proteins/enzymes present in the genome were downloaded from NCBI. FASTA le downloaded from NCBI was then uploaded into the RAST server to check the M. aeruginosa lysis and microcystin degradation pathways. CG viewer server database results showed a Circular representation of the Rhizobium sp. (AQ_MP) genome. From outward to inward: ORF (circles 1), CDS (circle 2&3), GC skew (circle 4), GC content (circle 5), ORF (circle 6), are shown in Figure 4. RAST server data results depict the presence of mlrC gene sequence from 184197 to 185678 in scaffold 2. Size of the sequence was found 1482bp and 494aa ( Figure 5). Some mlr (microcystin degrading gene) genes from NCBI were compared with conserved regions of Rhizobium sp. (AQ_MP), and match was observed and checked in pfam and InterProScan (Mitchell, A.L., et al., 2019, Bridge, A.J., et al., 2016. The result of InterProScan for the Rhizobium sp. (AQ_MP) conserved genome sequence is represented in Figure 5. At domain level, our target conserved genome sequence were classi ed under the protein family IPR009197 and domain IPR015995, IPR010799. This protein family was nearly related to mlrC domain. This family signi es the C-terminus of a bacterial gene cluster product that is related to the degradation of the toxin microcystin and is encoded in the mlr gene cluster. Phylogenetic tree of some mlrC sequence (NCBI) was compared with conserved sequences of Rhizobium sp. (AQ_MP) (Figure 6). Glycosyltransferase gene was also compared as outer protein family. It was seen that Rhizobium sp. (AQ_MP) conserved region was related to many Sphingopyxis sp. and Sphingomonas mlrC genes.
RAST server data exhibited the presence of siderophores in the Rhizobium sp. (AQ_MP) genome. Iron is a crucial element essential for key biotic processes. The common bacterial groups necessitate iron for existence and progression. Bioavailability ofIron is limited which is a persistent source of pressure in many biological structures. There is accruing indication that Fe restricts phytoplankton biomass in the equatorial Paci c (Wells, M.L., et al., 1994), the North Paci c gyre (Martin et al., 1989;Liu, Z.Z., et al., 2014), and the Southern Ocean (Helbling et al., 1991). For microorganisms obtaining iron is a signi cant challenge; capturing and integrating iron governs their existence. Cyanobacteria and algae, particularly responsible for biomass's primary production, requiresten times higher iron content than nonphotosynthetic prokaryotes (Brand, 1991). Bacteria, fungi, microalgae, and many higher plants have established speci c approaches for low iron bioavailability. Siderophores secretion is one of the approaches among them. Siderophores are the molecules that chelate iron with high a nity (Guerinor, 1994). Siderophores extracellularly solubilizes the iron from minerals of organic substances and transport them into cells when there is deprivation of iron. Photosynthesis and capturing light energy are closely related with photosynthetic pigments. In photosynthesis, iron plays a crucial role in chlorophyll-a production (Imai et al., 1999). Studies say cyanobacteria requires higher iron uptake that other algae (Brand, 1991). Liu, Z.Z., et al., 2014 has suggested that due to the presence of siderophores, photosynthetic pigment synthesis was inhibited in M. aeruginosa. In our study, Rhizobium sp. (AQ_MP) is connected to siderophore release, which indicates the inhibition of photosynthetic pigment synthesis. It could be responsible for the inhibition of M. aeruginosa growth due to the low bioavailability of iron (Martin, J.H., et al., 1989). In previous studies, it was observed that bacteria and cyanobacteria tend to compete in freshwater for the low bioavailability of iron (Liu, Z.Z., et al., 2014). Protein FASTA sequences of these loci were downloaded from NCBI and were submitted to Phyre software, and Pymol generates the structures of different siderophore and CAZymes (Supplementary Figure 3).
Nitrogen is a good source for the growth of cyanobacteria. Rhizobium sp. (AQ_MP) exhibited denitri cation activity against M. aeruginosa. The denitri cation pathway was present in the RAST server (Supplementary Figure 4). nar, nir, nos and nor are the genes responsible for the conversion of nitrate to ammonia (Tikariha, H. and Purohit, H.J., 2019) were also present in the genome. Some previously published work has been shown how signi cant denitri cation is to control algal blooms (Jiang, X., et al., 2020).
Zhang, H., Yu, Z., Huang, Q., Xiao, X., Wang, X., Zhang, F., Wang, X., Liu, Y. and Hu, C., 2011. Isolation, identi  Zhu, Y., Chen, P., Bao, Y., Men, Y., Zeng, Y., Yang, J., Sun, J. and Sun, Y., 2016. Complete genome sequence and transcriptomic analysis of a novel marine strain Bacillus weihaiensis reveals the mechanism of brown algae degradation. Scienti c reports, 6(1), pp.1-10.    Gene structure of annotated mlrC gene in Rhizobium. Protein structure of annotated mlrC conserved sequence was scanned through InterProScan tool to identify the multimodular domains of mlrC. (gst). This reaction occurs between the sulfhydryl group of the reduced glutathione and a nucleophilic center on the toxin. Mdha group is responsible for the nucleophilic center of microcystins. GSHconjugation is generally the rst sequence of the reaction, eventually producing an N-acetyl-cysteine which is mercapturic acid conjugate can be e ciently removed from the cell. Gammaglutamyltransferase (tgm) is responsible for the enzymatic cleaving of the γ-glutamic acid group of the