26371 Brazilian Journal of Development
Outlook of fosmid library from metagenomic of the symbionts associated with coral Siderastrea stellata: structural and functional screening for metabolic and antimicrobial activity
Perspectiva da biblioteca fosmidial a partir da metagenômica de simbiontes associados ao coral Siderastrea stellata: triagem estrutural e funcional da atividade metabólica e antimicrobiana
DOI:10.34117/bjdv6n5-188
Recebimento dos originais: 10/04/2020 Aceitação para publicação: 11/05/2020
Moara Silva Costa Doutora em Biologia e Biotecnologia de Microrganismos Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
Rachel Passos Rezende Doutora em Ciências Biológicas (Microbiologia) Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
Cristiane de Araújo Quinto Doutora em Biotecnologia Instituição: Universidade Estadual de Feira de Santana Endereço: Avenida Transnordestina, s/n - Novo Horizonte, Feira de Santana – BA. E-mail: [email protected]
Eric de Lima Silva Marques Doutor em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
Carlos Priminho Pirovani Doutor em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
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Bianca Mendes Maciel Doutora em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
Maria Clara Bessa Souza Graduanda em Biomedicina Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
João Carlos Teixeira Dias Doutor em Ciências Biológicas (Microbiologia) Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected]
ABSTRACT Microorganisms colonize the corals and produce compounds with interesting biological properties for biotechnology. The aim of the present study was to search for biocompounds with industrial potential through the cloning metagenomic DNA of symbionts the coral S. stellata in fosmid vector. The metagenomic library was analyzed using functional screening and sequencing via the Illumina MiSeq. Of the 3648 clones, eight were identified as proteolytic and six as amylolytic. The proteolytic clone, P07H3, also exhibited antimicrobial activity against S. aureus, S Enterica PT11, S Enterica PT4 and S Typhi. No clones were positive for lipase/esterase. Beta-lactamase was a single subsystem identified by Rapid Annotation using Subsystem Technology (RAST) through fosmid sequencing of clone P07H3. However, there were identifications of enzymes that participate in metabolic processes such as polysaccharide catabolism, oxidation-reduction, signal transduction and phosphorylation. When the hypothetical proteins were re-analyzed they exhibited a defined functional domain. All the identified open reading frames (ORFs) had low identity with proteins deposited in BLASTp. The sequencing shows that most of the genomic fragments of clone P07H3 possibly consist of new proteins. The Proteobacteria phylum had the greatest predominance among the analyzed fragments. This is the first report on the prospection of amylases, proteases and antimicrobials of coral S. stellata samples. The analyzes of this study help to improve the knowledge about the metabolic diversity of S. stellata still little explored and viable in the future study of the identified compounds.
Keywords: Biocompound; Cnidaria; Heterologous expression; Proteomics.
RESUMO Microrganismos colonizam os corais e produzem compostos com propriedades biológicas interessantes para a biotecnologia. O objetivo do presente estudo foi realizar buscas por biocompostos com potencial industrial através da clonagem de DNA metagenômico de simbiontes do coral S. stellata em vetor fosmídeo. A biblioteca metagenômica foi analisada usando triagem funcional e sequenciamento via Illumina MiSeq. Dos 3648 clones triados foram identificados oito clones proteolíticos e seis amilolíticos. O clone proteolítico P07H3 também apresentou atividade antimicrobiana contra S. aureus, S Enterica PT11, S Enterica PT4 and S Typhi. Não houve identificação de clones positivos para lipase/esterase. Beta-lactamase foi o único subsistema
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Palavras-chaves: Biocomposto; Cnidaria; Expressão heteróloga; Proteômica.
1 INTRODUCTION The Zooxanthellae coral Siderastrea stellata (Anthozoa: Scleractinia) is endemic to the Brazilian coast and occurs in shallow water environments and regions susceptible to intense maritime variations (Costa et al., 2001). Moreover, it is reportedly resistant to temperature changes, salinity, and turbidity of water (Leão et al., 2003). This metabolic plasticity together with high biological productivity makes this coral and its symbionts, a promising source in the search for new biomolecules. Only a small portion (0.001%) of microbial diversity in the marine environment is known (Amann et al., 1995). However, technological advances through studies that do not depend on the cultivation of microorganisms have provided knowledge of microbial taxonomic groups (Handelsman et al., 1998). Metagenomic tools with heterologous expression have enabled greater access to metabolic diversity. Through this approach, new metabolic routes, genes, and compounds are being discovered and applied to the understanding of various microbiomes and to the development of numerous biotechnological sectors (Dhanjal and Sharma 2018). Functional and structural metagenomics were used to detect biocompounds for biotechnological purposes. As such, the metagenomic DNA of the coral S. stellata microbiome was cloned in the fosmid vector pCC2FOS using a heterologous host. Samples of S. stellata DNA were tested for the first time to identify antimicrobial compounds and enzymes of economic interest.
2 MATERIAL AND METHODS 2.1 STUDY AREAS AND SAMPLING OF CORAL Siderastrea stellata Coral samples were collected in the Maraú peninsula BA (14º 06' 11" S, 39º 00' 53" W), certified by the National System For The Management of Genetic Heritage and Associated Traditional Knowledge (SisGen A7B90DA). Eight colonies with an approximate diameter of 10 cm were collected randomly in each coral S. stellata. The samples were transported on ice in a
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2.2 CONSTRUCTION OF THE METAGENOMIC LIBRARY OF CORAL MICROBIOME The metagenomic library was constructed according to the standardization of protocols already published in the literature (Wegley et al., 2007; Cheng et al., 2009). For total DNA extraction from the coral, 50g fragments of tissue, mucus, and skeleton were macerated with a mortar and pestle. The resulting macerate was diluted in sodium phosphate buffer (200 mM, pH 7.5) with 1% sodium pyrophosphate (Synth, São Paulo, Brazil) and 1% Tween® 80. The mixture was incubated under constant agitation at 180 g for 24 h at room temperature. The mixture was then centrifuged at 100 g for 5 min. The supernatant was then transferred to a falcon tube and re-subjected to centrifugation of 500 g for 10min. The pellet containing the microbial cells with 3g (wet weight) was used for DNA extraction with the E.Z.N.A.® Soil DNA kit (Omega bio-tek, Inc., Norcross, GA, USA) as per the guidelines of manufacturer. The metagenomic library was constructed using the CopyControl™ HTP Fosmid Library Production kit (Epicentre Biotechnologies, USA). The extracted metagenomic DNA was purified using electrophoresis at 1% agarose. The DNA fragments between 25 and 50 Kb were selected and their extremities were repaired. The fragments were bonded via vector pCC2FOS (Epicentre Biotechnologies, USA) followed by bundling of fosmids in phage lambda. The transformants of Escherichia coli EPI300-T1R were grown in Luria-Bertani (LB) medium with chloramphenicol 12.5 ug/ml at 37 °C overnight and stored with 20% glycerol. The enzyme activities of interest were analyzed based on stocks preserved at – 80 °C.
2.3 FUNCTIONAL ANALYSIS OF METAGENOMIC CLONES 2.3.1 Hydrolytic Screening Clones were screened for protease, amylase, and lipase/esterase enzyme activity using solid LB medium with added L - arabinose (0.01%), chloramphenicol (12.5 µg/mL) and 1% specific substrate. Skimmed milk were used as specific substrate for protease screening, starch was used for amylase screening, and tributyrin was used for lipase/esterase screenings. The clones were chopped on the surface of the medium and incubated at 37° C for 24h. Clones with positive activity were identified by detecting the transparent hydrolysis halo around the colony. To visualize positive clones in amylase screenings, 1 % iodine, and potassium iodide solution (KI:I2, 2:1 g/100mL of distilled H2O) was used (Lämmle et al., 2007). False positive results were eliminated using strains of non- transforming E. coli and by performing all functional screenings in triplicate.
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2.3.2 antimicrobial screening Four microbial strains were used in the antimicrobial trials: Staphylococcus aureus (INCQS 00186), Salmonella enterica subsp. enterica serovar Enteritidis phage type 4 (S. Enterica PT4), S. Enterica PT11 and S. Typhi (ATCC 5339). Microbes were cultivated in Muller-Hinton Agar medium (Acumedia, Lansing, Michigan) at 37° C for 16 h. The cultures were standardized by spectrophotometry using absorbance of 660 nm, OD of 0.135 in 0.9 % saline (Êxodo científica, Sumaré, São Paulo). These measurements are equivalent to 0.5 on the McFarland scale (~ 10-8 UFC/ mL). Each pathogenic strain was simply plated, inoculated in Muller-Hinton Agar medium. Small reservoirs of 6mm diameter were made in the center and 100 µl of supernatant of each positive clone was added. The plates were then incubated at 37°C for 16h. The positive microbial activity was visualized through detection of inhibition halo around the reservoir (Schillinger et al., 1989).
2.3.3 determination of enzymatic index and antimicrobial inhibition index The potential of the clones with positive activity detected through functional screening was evaluated using the enzymatic index and the antimicrobial inhibition index. The enzymatic index was determined using paquimeter through the mean diameter (cm) of the hydrolysis halos. The antimicrobial inhibition index was measured using the mean diameter (cm) of the inhibition halo. All enzymatic activities were measured in triplicate using Petri plates as experimental units.
2.4 FOSMID SEQUENCING OF CLONE P07H3 Clone P07H3 was sequenced via the Illumina MiSeq platform. The criterion of choice was the detection of the highest enzymatic index exhibited through hydrolytic screening and activity antimicrobial. Fosmid DNA was extracted according to the protocol described by (Sambrook, 1989). Extracted genetic material was processed according to the recommendations of the Nextera XT DNA Sample Preparation kit (Illumina, USA). The fosmid DNA was initially fragmented using the Amplicon Tagment Mix kit and the Tagment DNA Buffer at 55°C for 5 minutes. Then, the entire DNA was neutralized with Neutralize Tagment Buffer. The library was increased using index (7i and 5i) according to the specifications of the Nextera PCR Master Mix kit, purified with AMPure XP beads and standardized through qPCR, using the Kapa Library Quantification kit for Illumina sequencing platforms. Sequencing was carried out using MiSeq Reagent kit V3 (Illumina, USA) with 150 reading cycles in a MiSeq sequencer (Illumina, USA) at the State University of Santa Cruz (UESC), Ilhéus, BA.
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2.5 BIOINFORMATICS ANALYSIS The DNA reads obtained through sequencing were assembled in contigs (set of overlapping DNA segments) and subjected to pipeline Rapid Annotation using Subsystem Technology (RAST) (Aziz et al., 2008). Functional genes were selected to track metabolic activities. To functionally elucidate the sequenced fragments, basic local alignment search tool (BLASTx) (https://blast.ncbi.nlm.nih.gov/Blast.cgi.) was used with hierarchical classification against integrated bases such as the SEED subsystem (Overbeek et al., 2005) , the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa, 2002) and the Clusters of Orthologous Groups (COG) (Tatusov, 2000). Fragments compatible with the fosmid vector were disregarded for functional analyse. The protein sequences identified as hypothetical by SEED had their coding sequence (CDS) re- analyzed based on the database of the National Center for Biotechnology Information (NCBI). Amino acid sequence was predicted using the non-redundant protein (Nr) database with BLASTx and UniProtKB/Swiss-Prot. To determine genes of known function, we considered the best score/hit among the first 15 significant hits generated by the analysis. Duplicated fragments of potential protein-encoding genes (peg) and CDS were previously corrected and later used for predictions through functional analysis. Open reading frames (ORF) were identified using an ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/).
2.6 NUCLEOTIDE SEQUENCE ACCESSION NUMBER The fosmid insert sequence has been deposited in GenBank with accession number MN117929- MN117951.
3 RESULTS AND DISCUSSION 3.1 CONSTRUCTION AND FUNCTIONAL SCREENING OF THE METAGENOMIC LIBRARY OF CORAL S. stellata A fosmid based library of metagenomic DNA of S. stellata microbiome was constructed and obtained according to standards for the construct of fosmid libraries (Cheng et al., 2009; Wegley et al., 2007). With high molecular weight, above the band of the marker (36 Kb DNA Ladder, Invitrogen, São Paulo, Brazil), the extracted DNA showed inserts with approximately 40 Kb (Figure 1). A total of 3648 clones were obtained. Through the functional trials, eight with proteolytic activities (P07A8, P07H3, P08A1, P09A2, P16A3, P24A4, P25E6, and P38A6), and six clones with amylolytic activities (P15G4, P15G5, P30E1, P30H10, P35B5, and P35B7) were detected (Figure 2). Clone P07H3 also exhibited antimicrobial activity against all the test strains, demonstrating inhibitory potential against S. aureus, S. Enterica PT4, S. Enterica PT11 and S. Typhi (Figure 3). Antimicrobial
Braz. J. of Develop., Curitiba, v. 6, n. 5, p.26371-26392, may. 2020. ISSN 2525-8761 26377 Brazilian Journal of Development activity is well documented in Cnidaria, especially soft corals (Koh, 1997; Mariottini et al., 2016; Pereira et al., 2017). Others prospecting using corals samples also demonstrated the inhibitory action of defensive agents against S. aureus (ElAhwany et al., 2015; Qin et al., 2015), S. Enterica (Correa et al., 2011). Pereira et al., (2017), for example, tested 8 Brazilian coral species in different parts of the country and obtained an antimicrobial response against Serratia marcescens (with the highest inhibitory rates 83%), Vibrio parahaemolyticus, S. aureus, Bacillus cereus and Escherichia coli. No positive clone was obtained for the lipase/esterase trials. Until the present moment, no data has been published on the identification of proteolytic, amylolytic, or antimicrobial from samples of S. stellata. This fact increases the chances of capture, through biocompounds with characteristics never before prospected. The enzymatic indices and antimicrobial inhibition of the positive metagenomic clones from the present study are shown in Table1 and 2, respectively. The amylolytic clones obtained the best enzymatic index results with 2.57 cm.
Figure 1: Metagenomic DNA extracted from S. stellata coral. M1-100 ng do Fosmid Control DNA; M2- 50 ng do Fosmid Control DNA with approximately 50 ng; 1- Metagenomic DNA from S. stellata with repaired tips and approximate concentration of 40 ng/µl.
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Figure 2: Enzymatic screening of clones from the metagenomic library of S. stellata coral. a- Screening for protease with LB solid medium containing milk as substrate. Halo hydrolyzing the proteolytic clone P07H3. b- Screening for amylase in LB solid medium containing 1% starch as substrate and the production of amylase by two clones (P35B5 e P35B7).
Figure 3: Antimicrobial activity expressed by the metagenic clone P07H3 via inhibition halos against 4 bacterial strains: a- S Enterica PT11; b- S aureus INCQS 00186; c - S Typhi ATCC 5339 and d - S Enterica PT11. The red arrows indicate the wells that were fed with 100 μL of crude extract produced by the proteolytic clone P07H3.
Table 1: Enzymatic index of positive metagenomic clones of S. stellata
Enzymatic activity Metagenomic clone Enzymatic Index
Amylolytic P35B5 2.57
P15G4 2.48
P30H10 2.45
P15G5 2.17
P30E1 2.00 P35B7 1.63
Proteolytic P07H3 2.40
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P25E6 2.10
P38A6 1.80
P07A8 1.73
P08A1 0.93
P16A3 0.53
P24A4 0.43
P09A2 0.36
Table 2: Index of antimicrobial inhibition of metagenomic clone P07H3
Antimicrobian activity Inhibition Zone Index
S. aureus 3.35
S. Enterica PT11 2.60
S. Enterica PT4 2.20
S. Typhi 1.16
In contrast to other functional metagenomic studies, the results obtained here demonstrate higher index percentages for tested hydrolytic activity (Table 3). We correlated this higher mean of positive clone identification with the biological diversity inherent to coral reefs. Moreover, this result shows that, contrary to the findings of other studies (Daniel, 2005; Riesenfeld et al., 2004) the total sample of clones is not a prerequisite to obtain high metabolic representatives.
Table 3: Comparison among metagenomic libraries involving bioprospecting of biocatalysts
Enzymatic Sample Vector of Number Positive Screening Reference activity clones cloning total of efficiency % clones Amylase Coral Fosmid 3.648 6 0,16% This study
Human gut Fosmid 156.000 6 0,0038% (Tasse et al., 2010)
Marine sediment Fosmid 20.000 1 0,005% (Liu et al. 2012)
Soil Cosmid 350.000 1 0,0002% (Sharma et al., 2010)
Protease Coral Fosmid 3.648 8 0,219% This study
Sediment deep Fosmid 30.000 1 0,003% (Lee et al., 2007) sea
Soil Plasmid 100.000 1 0,001% (Gupta et al., 2002)
Soil of desert Plasmid 30.000 17 0,056% (Neveu et al., 2011)
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Failure to identify clones positive for lipase/esterase may be related to the rate of expression of these functional pathways. It is reported that for a gene to be detected by functional screening, its expression level should be high; thus, in the absence of high expression, a gene may not be detected (Daniel, 2005). In addition, a study of approximately 30 bacterial genomes using E. coli found that 60% of genes are not expressed (Gabor et al., 2004). The detection rate in some cases can be increased by using other host types such as Streptomyces, Pseudomonas (Wang et al., 2016), Rhizobium leguminosarum (Wexler et al., 2005), and various Proteobacteria (Craik et al., 2011). In another work, the non detection of clones with antimicrobial activity is attributed to the limitations of metagenomic techniques (Assis et al., 2014). This is because, in this same work, there were identifications of antimicrobial compounds from two isolates from samples of Atlantic forest soil through the classical techniques of microbial isolation.
3.2 FOSMID DNA SEQUENCING The genome of clone P07H3 analyzed using RAST had a size of 103,181 kb, 67 contigs, and 150 CDS with potential protein encoding genes – PEG. Most of the fragments were duplicated. These results demonstrate that the number of kb after excluding repeated sequences was almost the same as the fosmid vector size (40 kb). A single CDS was categorized in a subsystem with previously known predictions using RAST server (Figure 4). This was classified into the categories of virulence, disease, and defense, and the subcategories of resistance to antibiotics, toxic components, and beta- lactamase (EC 3.5.2.6) subsystem (PEG 144/CDS 91) (Table 4). The identified subsequence had a 98.86% similarity with beta-lactamase-producing S. enterica. It is highly probable that this fragment is related to the microbial defense system associated with coral S. stellata. The other 66 CDS did not fit into any subsystem. Of this total, five CDS were classified as non-hypothetical proteins (Table 4) and 61 CDS were classified as hypothetical proteins. Reanalysis of these sequences classified as hypothetical proteins after subjection to BLASTx showed a defined functional domain (Table 5). The biggest challenge of the post-genomic era is determining the protein function of genes (Sivashankari and Shanmughavel 2006) because the database used may not contain enough subsidies for comparative analyses, which prevents functional predictions.
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Figure 4: Functional representation of genes obtained by RAST from DNA sequencing of clone P07H3. The arrows indicate the only subsystem detected by SEED, KEGG and COG. This was classified in the beta-lactamase subsystem; category virulence, disease and defense; and subcategory resistance to antibiotics and toxic components.
Therefore, the observation that most of the pegs exhibited hypothetical or undefined function may be related to the metagenomic nature of the sample in question. As studies show, most genes prospected by metagenomics have an unknown function and are most probably involved in new functional systems (Ram et al., 2005). This indication corroborates the results of the present study, given that most of the gene sequences re-analyzed by BLASTx (Table 5), as well as the various ORFs identified (Table 6), had low identity (in %) in relation to other proteins available in the database. Furthermore, although some potential PEG (6 & 110) and ORFs (5 & 7) remained hypothetical proteins, after re-analysis, they exhibited defined domains. Identification of protein function based on the similarity of sequences is a classic method that helps elucidate unknown genes or genes without functional characterization. The LbR-like domain referring to the hypothetical protein of peg 6, for example, has a group of proteins with unknown functions with the domains UspA1, Neck, and YadA (Agnew et al., 2011). These domains are part of a class of pathogens and factors of virulence that act as adhesion molecules on the cell surface. YadA, for example, inhibits the complement activation pathway with the cell surface coating. Peg 110 also has a domain, alluding to bacterial adhesion cells (https://www.ncbi.nlm.nih.gov/Structure/cdd/pfam10633). This domain presented the NEW3 domain of alpha-galactosidase, which is associated with the NPCBM family (pfam08305) and is probably a new bonding module found in the N-terminal of glucosyl hydrolases (https://www.ncbi.nlm.nih.gov/Structure/cdd/pfam10633) (Naumoff, 2005). Although most of these sequences have not been fit into any subsystem, their respective functions are pertinent and interesting. The sequences, together with the identified ORFs (Table 6),
Braz. J. of Develop., Curitiba, v. 6, n. 5, p.26371-26392, may. 2020. ISSN 2525-8761 26382 Brazilian Journal of Development support the proteolytic and antimicrobial activities exhibited by clone P07H3. The correlation of these results proves that both cloned fragments are actively expressed and desirable for phenotypic and enzymatic characterization. Carboxypeptidase, beta-lactamase, isochorismatase, LbR-like, and CARB are some of the functional domains identified through fosmid sequencing and are compatible with proteolytic proteins and molecules with antimicrobial functions identified in functional screening. Another relevant domain from the biotechnological point of view is the CBM41 pullulanase superfamily corresponding to peg 25 (Guillén et al.; 2009). These enzymes are important in the starch industry, where they are used to hydrolyze amylopectin. Pullulanases consist of multiple distinct domains, including a catalytic domain belonging to the glycoside hydrolase (GH) family 13 and carbohydrate-binding modules (CBM), including CBM41 (Guillén et al.; 2009; Van Bueren and Boraston 2007; Domań-Pytka and Bardowski 2004; Vihinen and Mantsiila 1989). In total, seven ORFs were identified from the analyzed CDS (Table 6). They all exhibited low identity with proteins deposited in the databases used. The gene sequences constitute probable new proteins or proteins with unfamiliar functional characteristics. Based on the functional predictions, most of the ORFs have functional similarity to the protein sequences identified by RAST. Five ORFs were observed to have significant similarities to proteins from Proteobacteria. The other two ORFs identified have similarities with proteins from Firmicutes and unclassified Parcubacteria group. According to the different platforms adopted in the present study, the phylum Proteobacteria was the most predominant among the analyzed functional fragments. High indices of Proteobacteria representativity have already been observed in corals (Porporato et al., 2013; Pereira et al., 2017). However, the prevalence of this group is not restricted to corals since it also occurs in other marine organisms (González and Moran 1997; Webster et al., 2001).
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Table 4: Prediction of the non-hypothetical proteins of clone P07H3 metagenomic clone by RAST
Contigs/Peg Prediction Functional Host Organism Bacterial Division E-value Identity Similar Sequence
16/25 Bi-functional protein (secreted Bacillus halodurans C-125 Firmicutes 2e-10 27.04% fig|272558.1.peg.3697 alpha-amylase/dextrinase)
17/28 Probable oxidoreductase Shewanella sp. PV-4 Proteobacteria 2e-134 49.24% fig|323850.3.peg.511
194/34 Mobile element protein Salmonella enterica subsp. Proteobacteria 1.1e-012 71% fig|321314.4.peg.4524 enterica serovar Choleraesuis str. SC-B67
22/50 Sensory transduction histidine Shewanella oneidensis Proteobacteria 4e-48 46.81% fig|211586.1.peg.799 kinase MR-1
53/105 Butyrate kinase (EC 2.7.2.7) Geobacillus kaustophilus Firmicutes 3e-31 54.89% fig|235909.3.peg.1254 HTA426
91/144 Beta-lactamase Salmonella enterica Proteobacteria 2e-146 98.86% fig|321314.4.peg.4537
(EC 3.5.2.6)
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Table 5: Prediction of the hypothetical proteins of the metagenomic clone P07H3 obtained by RAST after reanalysis by BLASTx / NCBI
Protein Predictive Query Contig Peg Superfamily Putative microbial E-value Identity (%) Access number (Best hit) coverage (%)
NBD_sugar- Desulfobacterales bacterium 4 Butyrate kinase 98% 5,00E-33 64% OEU64046.1 kinase_HSP70_actin S5133MH16 2 5 Glycoside hydrolase ______Uncultured bacterium 83% 9,00E-16 100% AMO13185.1
Hipothetical protein 6 LbR-like Desulfobulbus sp. 58% 5,00E-42 65% RJX23626.1 C4563_01465
3 Polar amino acid transport system Desulfopila aestuarii DSM 7 ______95% 1,00E-05 54% SHO49813.1 substrate-binding 18488 protein
Thiotrichales bacterium 8 Isochorismatase ______96% 7,00E-29 81% KPK47378.1 SG8_50 5 Linear amide C-N 9 Ntn hidrolases Microbulbifer pacificus 70% 2,00E-06 56% WP_105103160.1 hydrolase
Full=Beta/alpha- 14 15 Alpha Amylase/AmyAc Paenibacillus polymyxa 48% 9,00E-04 27% P21543.1 amylase
Hypothetical protein Desulfobacterales bacterium 15 19 _____ 18% 3,00E-09 81% OEU79019.1 BA872_00860 C00003060
DUF488 domain- Desulfobacterium 18 30 DUF488 98% 1,00E-25 73% WP_015905209.1 containing protein autotrophicum
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GlcD/Cytokin- FAD-binding 31 bind/FAD_binding_4/B Vibrio parahaemolyticus 99% 3,00E-179 53% WP_021487001.1 oxidoreductase BE
Desulfobacteraceae bacterium 21 47 DDE Transposase Transposase DDE 88% 1,00E-24 74% PQP33224.1 SEEP-SAG10
22 51 Response regulator PRK15347/YesN/ REC Desulfobacteraceae bacterium 90% 5,00E-20 85% PNV83650.1
Fibronectin type III Parcubacteria group bacterium 26 65 ______96% 5,00E-18 30% KKQ67895.1 domain protein GW2011_GWA2_38_27
Paenibacillus popilliae ATCC 33 78 Beta-lactamase ______97% 2,00E-05 94% GAC44051.1 14706
34 79 Transporase IS1272 ______Staphylococcus auerus 98% 5,00E-23 94% CRE39519.1
106 ATP-binding protein Drf_FH1/TIP49 Desulfatitalea tepidiphila 96% 1,00E-40 65% WP_054032062.1 53 Hypothetical protein Desulfobacterales bacterium 110 CARDB 82% 1.3 37% OEU84654.1 BA865_14405 S5133MH4
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Table 6: Prediction of the ORFs of the fosmid insert of the metagenic clone P07H3
ORF Protein Family Host Organism Bacterial Division Query Cover E-value Identity Access
number
NBD_sugar- Desulfobacterales bacterium 1 Butyrate kinase Proteobacteria 98% 7,00E-106 69% OEU64046.1 kinase_HSP70_actin S5133MH16
Thiotrichales bacterium 2 Isochorismatase Cysteine hydrolase Proteobacteria 98% 4,00E-145 86% KPK47378.1 SG8_50
Parcubacteria group Unclassified Fibronectin type III Carboxipeptidase 3 bacterium Parcubacteria 86% 2,00E-17 32% KKQ67895.1 domain protein regulatory-like GW2011_GWA2_38_27 group
Alpha/amylase/X25_Ba Pul_like_Pullulan_Gpos/ 4 Beta/alpha-amylase Paenibacillus polymyxa Firmicutes 99% 3,00E-179 53% WP_021487001.1 CBM41_pululanase/CB M_20
Candidatus Desulfofervidus 5 Hypothetical protein Dockerin_like Proteobacteria 18% 3,00E-19 80% WP_066060804.1 auxilii
FAD-binding GlcD/FAD binding/ 6 Vibrio parahaemolyticus Proteobacteria 99% 3,00E-179 53% WP_021487001.1 oxidoreductase Cytokin-bind/
Hypothetical protein Desulfobacteraceae 7 BaeS Proteobacteria 75% 1,00E-176 78% PNV86248.1 C0610_07810 bacterium
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4 CONCLUSION The present study reveals the functional diversity and biological potential of the coral S. stellata and its bacterial community using a fosmid metagenomic library. Through Illumina sequencing, it was demonstrated that most genomic fragments of clone P07H3 consist of probable new proteins with biotechnological relevance. Most of these CDS were classified as hypothetical proteins and their ORFs exhibited low index percentages with proteins already deposited in the databases used. The following were identified among the non-hypothetical proteins predicted by RAST: beta-lactamase, alpha-amylase, probable oxidoreductase, sensory transduction of histidine kinase, transport of type ABC amino acids, periplasmatic component domain, and butyrate kinase. Carboxypeptidase, beta- lactamase, isochorismatase, LbR-like, and CARB are some of the functional domains identified through fosmid sequencing and are compatible with proteolytic and antimicrobial proteins. This work will assist the development of subsequent isolation and characterization methods for the identified potentially viable compounds.
ACKNOWLEDGMENTS Research supported by a grant from National Council for Scientific and Technological Development – CNPq. Process: 408442/20131. Moara Silva Costa’s doctorate grant was supported by a fellowship from the Bahia State Research Support Foundation (FAPESB). Process: 2544/2016.
DECLARATION OF COMPETING INTEREST The authors declare that there is no conflict of interest regarding the publication of this article.
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