New Multidrug Efflux Systems in a Microcystin-Degrading Bacterium Blastomonas fulva and Its Genomic Feature

A microcystin-degrading bacterial strain, Blastomonas fulva T2, was isolated from the culture of a microalgae Microcystis. The strain B. fulva T2 is Gram-stain-negative, non-motile, aerobic, non-spore-forming and phototrophic. The cells of B. fulva T2 are able to grow in ranges of temperature from 15 to 37 °C, with a pH of 6 to 8 and a salinity of 0 to 1% NaCl. Here, we sequenced the complete genome of B. fulva T2, aiming to better understand the evolutionary biology and the function of the genus Blastomonas at the molecular level. The complete genome of B. fulva T2 contained a circular chromosome (3,977,381 bp) with 64.3% GC content and a sizable plasmid (145.829 bp) with 60.7% GC content which comprises about 3.5% of the total genetic content. A total of 3842 coding genes, including 46 tRNAs and 6 rRNAs, were predicted in the genome. The genome contains genes for glycolysis, citric acid cycle, Entner–Doudoroff pathways, photoreaction center and bacteriochlorophylla synthesis. A 7.9 K gene cluster containing mlrA, mlrB, mlrC and mlrD1,2,3,4 of microcystin-degrading enzymes was identified. Notably, eight different efflux pumps categorized into RND, ABC and MFS types have been identified in the genome of strain T2. Our findings should provide new insights of the alternative reaction pathway as well as the enzymes which mediated the degradation of microcystin by bacteria, as well as the evolution, architectures, chemical mechanisms and physiological roles of the new bacterial multidrug efflux system.


Introduction
Hepatotoxic microcystins (MCs) are very stable against heat, pH, proteases and other hepatotoxic substances produced by Microcystis, which is the most well-known freshwater Cyanobacteria that causes harmful algal blooms [1][2][3][4]. MCs are cyclic heptapeptides structurally containing seven amino acids. MC variants contain different amino acid residues at two positions, which make the differentiation between variants of MCs. In MC-LR, two variable elements are leucine and arginine. MC-LR (microcystin-leucine-arginine) is most toxic among the MCs produced by Cyanobacteria. Due to their ring structure, MCs are highly resistant to degradation; however, they can be degraded by specific enzymes [2,[5][6][7][8].
The mlrA gene encodes a hydrolytic enzyme that opens the cyclic peptide of MC; the mlrB gene encodes metallopeptidase that degrades linearized MCs; the mlrC gene encodes an enzyme-like serine peptidase that breaks linearized MCs or oligopeptides; and the mlrD gene encodes a putative transporter protein involved in the active transport of MCs ( Figure 1).
Investigations tend to focus on microbial efflux systems that have direct clinical relevance for humans and animals. Efflux systems that are not clinically important can be uncovered with relative ease using Omics-technologies, but their actual activities and Investigations tend to focus on microbial efflux systems that have direct clinical relevance for humans and animals. Efflux systems that are not clinically important can be uncovered with relative ease using Omics-technologies, but their actual activities and significance in bacterial metabolism remain poorly understood and underexplored. As a result, research into new bacterial efflux pump systems and the proteins that cooperate with them is critical for understanding their evolution, architectures, molecular mechanisms and precise physiological roles.
Antimicrobial resistance is a global issue and raises questions regarding how to handle antimicrobials. Overusing antibiotics as growth promoters or preventives in humans and animals is one of the major causes of antibiotic resistance. Heavy metals and chemical fertilizers may drive environmental strains to express multidrug efflux pumps, resulting in cross-resistance [9][10][11][12]. In human and veterinary pathogenesis, bacterial multidrug efflux pumps defend microorganisms from antimicrobials. The physiological functions of bacterial multidrug efflux pumps can be described as follows: transport of antimicrobial peptides; protection against mammalian bile acids/salts and hormones; protection against plant-derived toxins; tolerance toward pH, salt, heavy metals and aromatic hydrocarbons; protection against oxidative and nitrosative stress; cell-to-cell signaling; bacterial biofilm formation, etc. [13][14][15][16][17][18][19][20]. Many, if not all, of these pumps perform physiological purposes other than protecting bacteria from antimicrobials [21][22][23][24]. Apart from antibiotics, efflux pumps can extrude a wide variety of non-antibiotic substrates, including heavy metals, organic compounds, plant-produced chemicals, quorum sensing signals and bacterial metabolites [25][26][27].
Blastomonas can grow aerobically, anaerobically or phototrophically [28][29][30][31][32][33]. The genome of this genus bears the puf genes, which code for proteins of the L and M subunits of the reaction center complex and LH1 complex [28,29,32,34]. Blastomonas species are Gram-negative, aerobic, non-spore-forming, reproduce via budding or asymmetric cell Antimicrobial resistance is a global issue and raises questions regarding how to handle antimicrobials. Overusing antibiotics as growth promoters or preventives in humans and animals is one of the major causes of antibiotic resistance. Heavy metals and chemical fertilizers may drive environmental strains to express multidrug efflux pumps, resulting in cross-resistance [9][10][11][12]. In human and veterinary pathogenesis, bacterial multidrug efflux pumps defend microorganisms from antimicrobials. The physiological functions of bacterial multidrug efflux pumps can be described as follows: transport of antimicrobial peptides; protection against mammalian bile acids/salts and hormones; protection against plant-derived toxins; tolerance toward pH, salt, heavy metals and aromatic hydrocarbons; protection against oxidative and nitrosative stress; cell-to-cell signaling; bacterial biofilm formation, etc. [13][14][15][16][17][18][19][20]. Many, if not all, of these pumps perform physiological purposes other than protecting bacteria from antimicrobials [21][22][23][24]. Apart from antibiotics, efflux pumps can extrude a wide variety of non-antibiotic substrates, including heavy metals, organic compounds, plant-produced chemicals, quorum sensing signals and bacterial metabolites [25][26][27].
Blastomonas can grow aerobically, anaerobically or phototrophically [28][29][30][31][32][33]. The genome of this genus bears the puf genes, which code for proteins of the L and M subunits of the reaction center complex and LH1 complex [28,29,32,34]. Blastomonas species are Gram-negative, aerobic, non-spore-forming, reproduce via budding or asymmetric cell division, generate carotenoids and bacteriochlorophylla and contain ubiquinone-10 as the primary respiratory quinone [28][29][30][31][32]. Thus far, two Blastomonas genomes have been analyzed and published [35,36]. In order to better understand its metabolism, we present the complete genome sequence of B. fulva T2, a microcystin-degrading strain with several multidrug efflux pumps isolated from a Microcystis culture. Our results focus on genes encoding proteins of major pathways of carbon metabolism, as well as genes related to photosynthesis, microcystin degradation and multidrug resistance system.

General Genomic Features
General features of strain B. fulva T2 were summarized in Table 1. The genome size of strain T2 was 4,123,210 bp with a DNA G+C value of 64.2 mol %, consisting of a single circular chromosome (CP020083) of 3,977,381 bp and a single circular plasmid (CP020084) of 145,829 bp ( Figure 2). Of the 3887 genes identified in the total genome, 3762 were proteinencoding genes; 6 were ribosomal, 46 were transfer RNAs and 4 were noncoding RNAs.  The third and fourth circles represent GC skew and G+C variation, respectively.

Carbon Metabolism and Phototrophic Related Genes
Genes encoding enzymes of a complete glycolysis and the citric acid cycle were discovered in B. fulva T2, as well as genes for the pentose phosphate and Entner-Doudoroff pathways. Like other Blastomonas members, the key genes of the Calvin-Benson cycle RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) are absent in the B. fulva T2. The anaplerotic CO 2 assimilation via the activity of phosphoenolpyruvate carboxykinase (pckA) is an important mechanism of non-autotrophic CO 2 fixation. Interestingly, the pckA (B5J99_17370) gene is present in the genome of strain T2. Members of the genus Blastomonas are characterized to produce bacteriochlorophylla and contain light-harvesting complexes. The genomes of strain T2 contain key photosynthesis genes encoding the light-harvesting protein beta and alpha subunits (pucBA) as well as reaction center L, M, C and H subunits (pufLM2C and puhA). PCR amplification also detected the puf genes, which code for proteins of the L and M subunits of the active photosynthetic reaction center and of the core light-harvesting complex ( Figure 3). Additionally, there are genes encoding for bacteriochlorophyll (bchFCXYZ) and light-independent protochlorophyllide reductase (chlLBN). Color key: pink, puf gene (photosynthetic reaction center subunits); puhA gene (photosynthetic reaction center H subunit): purple, puc gene (light-harvesting subunits); hyp1 gene (putative photosynthetic complex assembly protein): blue-gray, chl gene (light-independent protochlorophyllide reductase): green, bch genes (bacteriochlorophyll synthesis).

Microcystin Degradation and Related Genes
The results demonstrate that both total and extracellular microcystin concentrations were much lower compared to the control group ( Figure 4). The results indicate that the average degradation rate of MC-LR was 0.208 mg/L/d.

Microcystin Degradation and Related Genes
The results demonstrate that both total and extracellular microcystin concentrations were much lower compared to the control group ( Figure 4). The results indicate that the average degradation rate of MC-LR was 0.208 mg/L/d.

Microcystin Degradation and Related Genes
The results demonstrate that both total and extracellular microcystin concent were much lower compared to the control group ( Figure 4). The results indicate t average degradation rate of MC-LR was 0.208 mg/L/d. The significant finding was that B. fulva T2 could completely degrade MC-LR 4). To obtain unknown functional genes encoding enzymes responsible for M degradation, the mlrA, mlrB, mlrC and mlrD homolog genes of B. fulva T2 were analyzed through the genomic analysis. A genome-oriented study revealed that T2 was found to harbor homologs of the gene cluster mlrBD1,2,3,4AC (B5J99_03 B5J99_03490, Figure 5A), which were responsible for the conversion of MC-LR to [5]. The mlrA gene encodes a neutral metalloprotease with optimal function at  The significant finding was that B. fulva T2 could completely degrade MC-LR ( Figure 4). To obtain unknown functional genes encoding enzymes responsible for MC-LR degradation, the mlrA, mlrB, mlrC and mlrD homolog genes of B. fulva T2 were further analyzed through the genomic analysis. A genome-oriented study revealed that B. fulva T2 was found to harbor homologs of the gene cluster mlrBD 1,2,3,4 AC (B5J99_03460 to B5J99_03490, Figure 5A), which were responsible for the conversion of MC-LR to Adda [5]. The mlrA gene encodes a neutral metalloprotease with optimal function at pH 7.6 capable of the hydrolytic cleavage of the cyclic structure of microcystin, hence reducing its toxicity [37]. The sequences of the mlrA homolog gene (B5J99 03485) and 315 putatively translated amino acids have been determined (GenBank accession number ASR50645.1). The coding region of the mlrA gene had a G+C content of 65.9%. The nucleic acid and putative protein sequences analysis showed low similarities of the MlrA peptidase to mircocystin degrading enzymes ( Table 2). The alignment of these enzymes exhibits 37.9-42.2% amino acid sequence and 51.8-95.4% nucleic acid sequence similarities. Therefore, a phylogenetic analysis for the translated amino acid sequence of the mlrA homolog along with the microcystinase MlrA was performed, which is the first key degradative enzyme responsible for cleaving the cyclic MC into the linearized MC in the pathway of MC degradation ( Figure 5B). The phylogenetic tree showed that the mlrA gene formed a clade with the CPBP family (CAAX Proteases and Bacteriocin-Processing enzymes) intramembrane metalloproteases ( Figure 6). The MlrA as membrane protein belongs to the CPBP family converting cyclic MC to linear, and the analysis revealed that the gene may encodes putative microcystinase MlrA which breaks the conventional hydrogen bond to reduce its toxicity.     The mlrB gene (B5J99_03460) is located downstream of the mlrA and mlrD1,2,3,4 genes encoding proteins which cleave linear MC-LR to a tetrapeptide degradation product and possibly characterized as serine peptidase [5]. The mlrB gene sequence along with the putative translated amino acid sequence of 548 residues was determined (GenBank accession number ASR50640.1). The coding region had a G+C content of 68.2%. The pairwise analysis established low similarities of the MlrB peptidase to mirocystin degrading enzymes (Table 2). Pairwise alignment of these enzymes exhibits 27.1-31.3% amino acid sequence and a 49.0-49.9% nucleic acid sequence similarities.
The mlrC gene (B5J99_03490) encoding a metallopeptidase MlrC is located next to the mlrA homolog and responsible for the hydrolysis of Adda-Glu in the degradation o tetrapeptide to Adda [8], and a similar gene organization was observed in genomes o Sphingopyxis sp. C-1 and Sphingosinicella microcystinivorans B9 ( Figure 5A) [38,39]. Like mlrB, the mlrC gene is transcribed in the opposite direction to the mlrA and mlrD genes ( Figure 5A). The mlrC gene (B5J99_03490) sequence along with the putative translated amino acid sequence of 278 residues was determined (GenBank accession number The mlrB gene (B5J99_03460) is located downstream of the mlrA and mlrD 1,2,3,4 genes encoding proteins which cleave linear MC-LR to a tetrapeptide degradation product and possibly characterized as serine peptidase [5]. The mlrB gene sequence along with the putative translated amino acid sequence of 548 residues was determined (GenBank accession number ASR50640.1). The coding region had a G+C content of 68.2%. The pairwise analysis established low similarities of the MlrB peptidase to mirocystin-degrading enzymes ( Table 2). Pairwise alignment of these enzymes exhibits 27.1-31.3% amino acid sequence and a 49.0-49.9% nucleic acid sequence similarities.
The mlrC gene (B5J99_03490) encoding a metallopeptidase MlrC is located next to the mlrA homolog and responsible for the hydrolysis of Adda-Glu in the degradation of tetrapeptide to Adda [8], and a similar gene organization was observed in genomes of Sphingopyxis sp. C-1 and Sphingosinicella microcystinivorans B9 ( Figure 5A) [38,39]. Like mlrB, the mlrC gene is transcribed in the opposite direction to the mlrA and mlrD genes ( Figure 5A). The mlrC gene (B5J99_03490) sequence along with the putative translated amino acid sequence of 278 residues was determined (GenBank accession number ASR50646.1), and the coding region had a G+C content of 65.1%. The predicted proteins exhibited no significant matches with previously characterized MlrC, but the enzyme was identified as aminopeptidase, which may functionally work the same way as mlrC.
Previously, Bourne et al. (2001) showed that the function of mlrD is unclear [5]. It was postulated that the mlrD gene was involved in the transfer of either parent MC into the cellular environment or MC degradation products out of the cell. Within the 7.9 kb gene cluster, four mlrD gene homologs (B5J99_03465-03480) were identified just downstream of mlrA, and this is not an usual gene structure previously observed. The sequences of nucleic acid and amino acid were applied at the NCBI database using the BLASTN and BLASTP program. As a result, high sequence similarity suggested that the proteins belong to the ABC-type dipeptide/oligopeptide/nickel transport system as permease components. This family of proteins is involved in the transport and metabolism of amino acids and inorganic ions. As with mlrC, there were no significant matches between the predicted proteins and previously identified mlrD genes in the database. However, the function of the putative proteins was identical to that of microcystinase MlrD, so it is possible that one of them acts as MlrD or that all four genes work together to complete their functions.
Strain T2, which has exhibited a significant capacity for microcystin degradation, possesses a cluster of mlr gene homologs. It is considered to encode different hydrolytic proteins potentially involved in the initial or intermediate steps of MC degradation. The enzymes MlrA, MlrB and MlrC possibly undertake aminolysis processes, whereas the MlrD proteins intake or uptake small peptides that are produced. To date, the whole genomic sequences of only five MC-LR-degrading bacterial strains, namely Novosphingobium sp. THN-1, Novosphingobium sp. MD-1, Sphingopyxis sp. C-1, Sphingopyxis sp. YF1 and Sphingosinicella microcystinivorans B9 have been obtained [38][39][40][41][42]. Moreover, there are rare reports about functional genes for MC degradation with the exception of the mlrBDAC gene cluster. Although Okano et al. reported that Novosphingobium MD-1 possesses mlrE and mlrF genes participating in MC degradation, further research is needed to assess the MC-degrading function and gene structures. Searching novel mlr gene homologs help scientists better understand their functional characteristics and metabolic pathways. Intriguingly, PCR studies failed to amplify genes encoding hydrolytic enzyme (mlrA), metallopeptidase (mlrB), suspected serine peptidase (mlrC) and putative transporter protein (mlrD), suggesting that unique enzymes or a pathway for MC-LR might exist in this strain.

Multidrug Efflux Systems
Bacterial efflux pumps are classified into seven distinct families or superfamilies: (I) ABC, the ATP-binding cassette superfamily; (II) RND, resistance-nodulation−celldivision superfamily; (III) MFS, the major facilitator superfamily; (IV) MATE, the multidrug and toxic compound extrusion family; (V) DMT, the drug/metabolite transporter superfamily; (VI) PACE, the proteobacterial antimicrobial compound efflux family; and (VII) AbgT, the p-aminobenzoyl-glutamate transporter family [43][44][45][46][47][48]. The quantity and type of efflux pumps vary significantly in bacterial lineages. A critical distinction in this regard is between Gram-positive and Gram-negative bacteria, which have fundamentally different cell envelope architectures and hence different requirements and capacities for small molecule export [49]. Members of the RND, ABC and MFS superfamilies have been shown to form tripartite complexes with periplasmic adapter proteins and outer-membrane proteins in Gram-negative bacteria, facilitating substrate efflux through the outer membrane [43,[45][46][47][48]. Eight different efflux pumps have been discovered in the genome of strain T2 (Figure 7), including two CmeABC pumps, two AcrAB-TolC pumps and one CzcABC pump of the RND type; one MacAB-TolC pump and one HylBD-OMP pump of the ABC type; and one MFS-HylD-OMP pump of the MFS type.  Three-gene operon cmeABC is a tripartite efflux system containing the fusion prot CmeA, the inner membrane protein CmeB and the outer membrane protein CmeC, wh confers resistance to a range of antibiotics, heavy metals, bile salts and other antimicrob agents [50]. Two cmeABC gene clusters consisting of cmeA1 (B5J99_10820), cm (B5J99_15890), cmeB1 (B5J99_10825), cmeB2 (B5J99_15895), cmeC1 (B5J99_10830) and cm (B5J99_15900) genes were found in the genome of B. fulva T2 (Figure 7). Genomic analy of strain B. fulva T2 revealed the presence of two putative cmeABC efflux pump oper closely homologous to that of Sphingomonas wittichii RW1 [51]. This locus contains cmeA1 gene putatively encoding the fusion protein functioning as the periplasmic adap protein CmeA showed 59.3% of similarity (48.7% for cmeA2) to that of S. wittichii RW1, cmeB1 gene encoding the inner-membrane RND protein CmeB showed 62.5% of simila (58.1% for cmeB2) to S. wittichii RW1 followed by the cmeC1 gene encoding ou membrane CmeC which showed 50.4% of similarity (44.8% for cmeC2) to that of S. witti RW1 (Table 3). Phylogenetic studies of CmeABC efflux proteins showed that cmeA homolog genes formed clades with members of efflux fusion protein, inner membra protein and outer membrane protein, respectively. Based on their placement in cla with cmeABC ( Figure 8) and similarities with related proteins, it is likely that strain B. fu T2 possesses two CmeABC pumps.

RND Type CmeABC Efflux System
Three-gene operon cmeABC is a tripartite efflux system containing the fusion protein CmeA, the inner membrane protein CmeB and the outer membrane protein CmeC, which confers resistance to a range of antibiotics, heavy metals, bile salts and other antimicrobial agents [50]. Two cmeABC gene clusters consisting of cmeA 1 (B5J99_10820), cmeA 2 (B5J99_15890), cmeB 1 (B5J99_10825), cmeB 2 (B5J99_15895), cmeC 1 (B5J99_10830) and cmeC 2 (B5J99_15900) genes were found in the genome of B. fulva T2 (Figure 7). Genomic analysis of strain B. fulva T2 revealed the presence of two putative cmeABC efflux pump operons closely homologous to that of Sphingomonas wittichii RW1 [51]. This locus contains the cmeA 1 gene putatively encoding the fusion protein functioning as the periplasmic adaptor protein CmeA showed 59.3% of similarity (48.7% for cmeA 2 ) to that of S. wittichii RW1, the cmeB 1 gene encoding the inner-membrane RND protein CmeB showed 62.5% of similarity (58.1% for cmeB 2 ) to S. wittichii RW1 followed by the cmeC 1 gene encoding outer-membrane CmeC which showed 50.4% of similarity (44.8% for cmeC 2 ) to that of S. wittichii RW1 (Table 3). Phylogenetic studies of CmeABC efflux proteins showed that cmeABC homolog genes formed clades with members of efflux fusion protein, inner membrane protein and outer membrane protein, respectively. Based on their placement in clades with cmeABC ( Figure 8) and similarities with related proteins, it is likely that strain B. fulva T2 possesses two CmeABC pumps.

AcrAB-TolC Efflux System
The AcrAB-TolC efflux system is known to be responsible for the extrusion of a wide variety of compounds in a number of Gram-negative bacteria, such as E. coli, Salmonella, Klebsiella, Erwinia and Acinetobacter. These compounds include antibiotics, lipophilic antimicrobial drugs, dyes, detergents and organic solvents [52]. The genome of strain T2 has been found to possess complete genes for two AcrAB-TolC efflux pumps, which are resistant to chloramphenicol, fluoroquinolone, tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid and β-lactam antibiotics [53]. Five genes acrA1 (B5J99_04740), acrA2 (B5J99_18545), acrB1 (B5J99_04745), acrB2 (B5J99_18540) and acrA3 (B5J99_10590) encoding for inner-membrane and fusion proteins were observed, and these five genes along with the tolC gene which encodes outer-membrane protein make two AcrAB-TolC pumps (Figure 7). Genomic analysis revealed the presence of two putative acrAB efflux pump operons closely homologous to that of Rhodospirillum centenum SW, Caulobacter crescentus CB15 and Erythrobacter litoralis HTCC2594 [54][55][56]. The acrA1 gene putatively encoding the

AcrAB-TolC Efflux System
The AcrAB-TolC efflux system is known to be responsible for the extrusion of a wide variety of compounds in a number of Gram-negative bacteria, such as E. coli, Salmonella, Klebsiella, Erwinia and Acinetobacter. These compounds include antibiotics, lipophilic antimicrobial drugs, dyes, detergents and organic solvents [52]. The genome of strain T2 has been found to possess complete genes for two AcrAB-TolC efflux pumps, which are resistant to chloramphenicol, fluoroquinolone, tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid and β-lactam antibiotics [53]. Five genes acrA 1 (B5J99_04740), acrA 2 (B5J99_18545), acrB 1 (B5J99_04745), acrB 2 (B5J99_18540) and acrA 3 (B5J99_10590) encoding for inner-membrane and fusion proteins were observed, and these five genes along with the tolC gene which encodes outer-membrane protein make two AcrAB-TolC pumps (Figure 7). Genomic analysis revealed the presence of two putative acrAB efflux pump operons closely homologous to that of Rhodospirillum centenum SW, Caulobacter crescentus CB15 and Erythrobacter litoralis HTCC2594 [54][55][56]. The acrA 1 gene putatively encoding the acriflavin resistance protein A precursor working as the periplasmic adaptor protein AcrA had a 35.1% similarity to the membrane-fusion protein of R. centenum SW. Additionally, the acrB1 gene encoding the acriflavin resistance protein B residing as the inner membrane RND protein AcrB displayed a similarity of 50.1% to that of C. cres-centus CB15. Another AcrAB pump was also identified, where the acrA 2 gene had 56.0% of similarity to that of E. litoralis HTCC2594, and acrB 2 had 72.9% of similarity to that of E. litoralis HTCC2594 (Table 3). Phylogenetic studies of AcrAB efflux proteins have revealed that acrAB homolog genes formed clades with members of efflux fusion proteins and inner membrane proteins, respectively. In this efflux system, TolC serves as an outer membrane protein which interacts with inner membrane efflux proteins to expel antibiotics or export virulence factors from bacteria [57]. The tolC gene also works in combination with other RND, ABC and MFS efflux pumps [58,59], and the genome of strain T2 possesses two tolC genes encoding outer-membrane proteins, presumably working cooperatively as outer-membrane proteins of AcrAB-TolC, MacAB-TolC and other two pumps. Based on their placement in clades with acrAB ( Figure 9) and similarities with related proteins, strain B. fulva T2 is likely to possess two AcrAB-TolC pumps.
Mol. Sci. 2022, 23, x FOR PEER REVIEW and MFS efflux pumps [58,59], and the genome of strain T2 possesses tw encoding outer-membrane proteins, presumably working cooperative membrane proteins of AcrAB-TolC, MacAB-TolC and other two pumps. B placement in clades with acrAB ( Figure 9) and similarities with related pro fulva T2 is likely to possess two AcrAB-TolC pumps.

CzcABC Efflux System
Microorganisms may be exposed to a variety of exogenous environm such as hydrocarbons and heavy metals, which may be of natural or anthrop and may be harmful if allowed to accumulate in bacterial cells [60]. Efflu key roles in the removal of these substrates. CzcCBA belongs to the family o efflux (HME) RND pumps and are involved mainly in response to the expo

CzcABC Efflux System
Microorganisms may be exposed to a variety of exogenous environmental toxins, such as hydrocarbons and heavy metals, which may be of natural or anthropogenic origin and may be harmful if allowed to accumulate in bacterial cells [60]. Efflux pumps play key roles in the removal of these substrates. CzcCBA belongs to the family of heavy metal efflux (HME) RND pumps and are involved mainly in response to the export of Co 2+ , Zn 2+ and Cd 2+ (czc) [61]. Three-gene operon czcCBA is also a tripartite efflux system consisting of the three proteins CzcC, CzcB and CzcA. The genome possesses the czcC gene (B5J99_01260) that encodes the heavy metal RND efflux outer-membrane protein CzcC, the acrB gene (B5J99_01265) encoding metal cation efflux fusion protein CzcB and the czcA (B5J99_01270) encoding for heavy metal RND efflux inner-membrane protein CzcA, which form a CzcCBA pump (Figure 7). Comparative genomic analysis revealed that this putative czcCBA efflux pump operon is closely homologous to that of Caulobacter crescentus CB15 [55], where the czcC gene had 37.0% of similarity to the cobalt-zinc-cadmium resistance gene of C. crescentus CB15, the czcB showed 56.4% of similarity to that of C. crescentus CB15 and the czcA gene showed 71.3% of similarity to that of C. crescentus CB15 (Table 3). Phylogenetic studies of CzcCBA efflux proteins have revealed that czcCBA homolog genes formed clades with members of efflux fusion proteins and inner-membrane proteins, respectively. Based on their placement in clades with czcCBA ( Figure 10) and similarities with related proteins, the strain B. fulva T2 is likely to possess a CzcCBA pump.

ABC Type
ABC transporters are a family of membrane proteins that mediate different driven transport activities. Transporters classified within the ABC superfamil ubiquitous to all domains of life and are likely to be the most abundant superfam transport proteins on Earth [62]. Transporters belonging to this family are known

ABC Type
ABC transporters are a family of membrane proteins that mediate different ATP-driven transport activities. Transporters classified within the ABC superfamily are ubiquitous to all domains of life and are likely to be the most abundant superfamily of transport proteins on Earth [62]. Transporters belonging to this family are known to be responsible for uptake or efflux of substrates like vitamins, amino acids, lipids, peptides, ions and drugs. Two ABC-type efflux pumps, namely MacAB-TolC and HlyBD-OMP, are present in the genome of B. fulva T2 (Figure 7). The MacAB-TolC pump is known to have resistance to a variety of macrolides, aminoglycosides and polymyxins [63].

MacAB-TolC Efflux System
Here, the macB gene encodes for an ABC superfamily half-transporter, which combines with the MacA periplasmic fusion protein that binds to the outer-membrane channel TolC. The macB gene (B5J99_15995) encodes the macrolide export ATP-binding protein MacB, and the putative protein sequences exhibits 46.4% of amino acid sequence similarity to Rhodospirillum centenum SW [54]. The macA gene (B5J99_16005) encoding periplasmic adapter protein MacA shared 43.4% of sequence similarity with that of Geobacter sulfurreducens PCA [64]. A ftsE gene was also identified (Figure 7), encoding a cell-division signaling protein that is part of the cytoplasmic ATP-binding component MacB. This gene shared 61.6% of similarity with the ftsE gene of Geobacter uraniireducens Rf4 (CP000698). In this efflux system, TolC acts as an outer-membrane protein to form the efflux pump. The phylogenetic analysis for the MacBA homolog associated with the amino acid sequence of the macrolide export proteins was performed ( Figure 11). The MacBA homolog forms a clade with related proteins and likely possesses a MacBA-TolC pump.

HlyBD 2 -OMP 2 Efflux System
Another ABC-type tripartite pump HlyBD 2 -OMP 2 was detected in the genome of B. fulva T2 (Figure 11). The gene hlyB (B5J99_15940) encoding putative inner-membrane protein HlyB, which acts in concert with the adaptor HlyD to export the large protein toxin, hemolysin from the cytoplasm across both membranes in a concerted step, had 51.8% of similarity to the toxin secretion ABC transporter protein of Novosphingobium aromaticivorans DSM 12444 (CP000248); hlyD 2 (B5J99_15945), which encodes the RND efflux membrane fusion protein, had 57.5% of similarity to that of N. aromaticivorans DSM 12444; omp 2 (B5J99_15950), which encodes the outer-membrane protein had 48.1% of similarity to that of N. aromaticivorans DSM 12444 (Table 3). Phylogenetic studies of HlyBD 2 -OMP 2 efflux proteins have revealed that hlyBD-omp 2 homolog genes formed clades with members of efflux fusion proteins and inner membrane proteins, respectively. The phylogenetic analysis for the HlyBD homolog associated with the amino acid sequence revealed that the homologs form a clade with related proteins and likely possesses a HlyBD 2 -OMP 2 pump.

MFS Type: MFS-HlyD-OMP Efflux System
MFS is also known to form tripartite complexes with periplasmic fusion proteins and outer-membrane proteins to facilitate substrate efflux across the outer membrane. A tripartite pump MFS 1 -HlyD 1 -OMP 1 was also observed in the genome of B. fulva T2 ( Figure 12). This gene cluster includes the MFS 1 gene (B5J99_05315) putatively encoding the inner membrane protein MFS transporter protein locating at inner membrane, hlyD 1 (B5J99_05310) that encodes HlyD family secretion protein as fusion protein and omp 1 that encodes the outer-membrane protein. A TetR family regulator gene TetR was present just upstream of the operon, downregulating the pump expression. The genomic analysis revealed the presence of the MFS 1 -HlyD 1 -OMP 1 efflux pump operon closely homologous to that of Sphingomonas wittichii RW1 [51]. The comparative analysis showed that the gene MFS 1 had 39.5% of similarity to that of the S. wittichii RW1, hlyD 1 and omp 1 gene which had 54.9% and 53.2% of similarity to that of S. wittichii RW1, respectively (Table 3). Phylogenetic analysis of MFS efflux proteins revealed that all three genes formed clades with members of efflux fusion proteins and inner-membrane proteins, respectively. Based on their placement in clades with related proteins, the strain B. fulva T2 probably possesses an MFS-type pump.
Bacteria and other microorganisms have developed the ability to mediate the efflux of small molecule substrates and ions. Efflux pumps are able to transport diverse small molecules out of the cell. Transporters in Gram-negative bacteria typically have the broadest substrate recognition profiles, and it is conceivable that these pumps in particular could recognize and transport substrates such as amino acids and other metabolites. This analysis reveals that the genome of B. fulva T2 encodes a variety of transport proteins required for the export of toxic compounds and the import of essential molecules such as sugars, amino acids, ions and peptides. The classification of transporters into categories such as RND, ABC or MFS transporters does not necessarily indicate anything about their function in vivo. However, molecular genetics is useful for gaining initial insights into the significance of transporters to bacterial physiology. In addition, a comprehensive biochemical analysis is required to completely understand the biological roles of RND-, ABC-and MFS-type transporters and other transport systems, as well as to determine their action mechanisms.
Phylogenetic analysis of MFS efflux proteins revealed that all three genes formed cla with members of efflux fusion proteins and inner-membrane proteins, respectively. Ba on their placement in clades with related proteins, the strain B. fulva T2 probably posses an MFS-type pump. Bacteria and other microorganisms have developed the ability to mediate the eff of small molecule substrates and ions. Efflux pumps are able to transport diverse sm molecules out of the cell. Transporters in Gram-negative bacteria typically have broadest substrate recognition profiles, and it is conceivable that these pumps particular could recognize and transport substrates such as amino acids and ot metabolites. This analysis reveals that the genome of B. fulva T2 encodes a variety transport proteins required for the export of toxic compounds and the import of essen molecules such as sugars, amino acids, ions and peptides. The classification transporters into categories such as RND, ABC or MFS transporters does not necessa indicate anything about their function in vivo. However, molecular genetics is useful gaining initial insights into the significance of transporters to bacterial physiology

Isolation and Culture Conditions
Blastomonas fulva T2 was isolated from a Microcystis culture in Daejeon, Republic of Korea, using dilutions to extinction (10 6 or 10 7 ) method in R2A medium (Difco, Franklin Lakes, NJ, USA) at 25 • C for 7 days. Microcystis cells were grown in 500 mL standard cell culture flasks using Blue-Green (BG11) broth (Merck, St. Louis, MO, USA) under the following conditions: 20 • C, 35% humidity, 12 h:12 h light-dark photoperiod, 20 µmol m −2 s −1 irradiance and 200 rpm agitation. A 100 L subsample of the suspended material from the Microcystis culture was aseptically disseminated onto R2A agar under heterotrophic conditions. The isolated strain T2 then was routinely sub-cultivated on R2A agar at 30 • C for 48 h and kept in a glycerol solution (20%, v/v) at −70 • C for long-term preservation.

Phylogenetic and Genomic Analyses
Genomic DNA was extracted using the FastDNA TM SPIN DNA-extraction kit according to the manufacturer's instructions and purity was checked using a ND2000 spectrometer (Nanodrop Technologies, Inc., Wilmington, DE, USA). PCR amplification of the pufLM genes was performed following the method previously described [32,34]. Genomic sequencing was performed using the PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA) and the Illumina HiSeq platform at Macrogen (Seoul, Korea). SMRT Link v5.0.1 was used to do the sequence quality control of reads filtration and the assemblage [65]. The genome was annotated and compared in the RAST pipeline and the SEED Viewer, respectively [66,67], where protein functions were defined in the FIGfam collection [68]. The predicted protein coding sequences (CDSs) were compared to the COGs (Clusters of Orthologous Groups) database (http://www.ncbi.nlm.nih.gov/COG/, May 2022) to determine the functional category and summary statistics [69,70]. Phylogenetic analysis of protein sequences was performed using MEGA 7.0 [71]. Amino acid sequence alignment was done using the programs CLUSTAL X (version 1.8) [72], and the phylogenetic trees were reconstructed using the algorithm of minimum-evolution (ME) [73]. Bootstrap values of the phylogenetic trees were calculated on 1000 resamplings of the sequences [74]. For trees of mlr-related and multidrug efflux system proteins, the amino acid sequences were used as queries in BLASTP searches to identify other homologs.

Microcystin Assay
For MC degrading assays, strain T2 was cultivated in 500 mL culture flasks in R2A medium containing 20 µg·L −1 MC-LR (Supelco, Belfort, PA, USA) with constant shaking at 150 rpm at 30 • C for 24 h [75]. The cell growth was measured at OD 600 nm and cell culture was harvested at 12 h and 24 h. The supernatant was filtered through a 0.22 µm polycarbonate filter after 10 min of centrifugation at 10,000× g. The MC was quantified using QuantiPlate™ Kit (Envirologix, Inc., Portland, ME, USA). All experiments were performed in triplicate.

Conclusions
The genome of B. fulva T2 represents the first detailed analysis of a genome from a species of Blastomonas that grows optimally at moderate temperatures and neutral pH. To summarize our investigations, the strain T2 from a Microcystis culture belonging to the genus Blastomonas was studied through genomic analysis, which confirmed its capability to degrade MC-LR. A 7.9K gene cluster containing mlrA, mlrB, mlrC and mlrD 1,2,3,4 genes is involved in the degradation of microcystin. Like other members in the genus Blastomonas, phototrophic systems were detected. B. fulva T2 also contains genes encoding for proteins of RND-, ABC-and MFS-type multidrug efflux systems. Several efflux pumps have been identified in a single bacterial cell, and multiple efflux pumps may have additive or greaterthan-additive effects on drug resistance and substrate transport. The analysis of genes and their function is challenging, because it is frequently impeded by protein preparations necessary for in vitro experiments or the determination of the three-dimensional structure of transporters. Pathogenic and nonpathogenic bacterial genomes contain multiple uncharacterized transporters that may be essential for the growth and/or survival of these organisms and should therefore not be disregarded. The genome sequence and comparative genome analyses of B. fulva T2 provide a genetic blueprint and physiological characteristics which help us to understand the different metabolism and evolutionary features of the genus Blastomonas, especially the multidrug efflux system in non-pathogenic bacteria.  Data Availability Statement: The complete genome sequence of Blastomonas fulva T2 has been deposited at GenBank under the accession number CP020083 (CP020084 for plasmid). The strain is available from two different culture collections, namely JCM (Japan Collection of Microorganisms) and KCTC (Korean Collection for Type Cultures), with the accession numbers KCTC 42354 T and JCM 30467 T , respectively.

Conflicts of Interest:
The authors here declare that they have no known competing financial interest or personal relationships that could have appeared to have any influence.