Genome Sequences of Serratia Strains Revealed Common Genes in Both Serratomolides Gene Clusters

biology Article Genome Sequences of Serratia Strains Revealed Common Genes in Both Serratomolides Gene Clusters Catarina Marques-Pereira †, Diogo Neves Proença * and Paula V. Morais Department of Life Sciences, Centre for Mechanical Engineering, Materials and Processes, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal; [email protected] (C.M.-P.); [email protected] (P.V.M.) *Correspondence: [email protected]; Tel.: +351-239240798 †Current address: Center for Neuroscience and Cell Biology & Center for Innovative Biomedicine and Biotechnology, University of Coimbra, 3004-504 Coimbra, Portugal. Received: 31 October 2020; Accepted: 17 December 2020; Published: 20 December 2020   Simple Summary: Biosurfactants are amphiphilic molecules produced by microorganisms with a hydrophilic and a hydrophobic group, able to reduce surface tension. These molecules are largely used in the environmental, food, pharmaceutical, medical, and cleaning industries, among others. Serratia strains are ubiquitous microorganisms with the ability to produce biosurfactants, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi. This work used comparative genomics to determine the distribution and organization of the serrawettins W1 and W2 biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW and serrawettin W2 biosynthetic gene swrA were present in 17 and 11 Serratia genomes, respectively. The same genes in the biosynthetic clusters frame the swrW and swrA biosynthetic genes. This work identified four genes common to all serrawettin gene clusters, highlighting their key potential in the serrawettins biosynthetic process. Abstract: Serratia strains are ubiquitous microorganisms with the ability to produce serratomolides, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi and may have a nematicidal activity against phytopathogenic nematodes. Serrawettins W1 and W2 from different strains have different structures that might be correlated with distinct genomic organizations. This work used comparative genomics to determine the distribution and the organization of the serrawettins biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. The serrawettin W1 and W2 gene clusters’ organization was established using antiSMASH software and compared with single and short data previously described for YD25 T Serratia. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW was present in 17 Serratia genomes. Eighty different coding sequence (CDS) were assigned to the W1 gene cluster, 13 being common to all clusters. The serrawettin W2 swrA gene was present in 11 Serratia genomes. The W2 gene clusters included 68 CDS with 24 present in all the clusters. The genomic analysis showed the swrA gene constitutes five modules, four with three domains and one with four domains, while the swrW gene constitutes one module with four domains. This work identified four genes common to all serrawettin gene clusters, highlighting their essential potential in the serrawettins biosynthetic process. Keywords: Serratia; genome; serrawettin; biosynthetic gene cluster; serratomolides; swrW gene; swrA gene Biology 2020,9, 482; doi:10.3390/biology9120482 www.mdpi.com/journal/biology Biology 2020,9, 482 2 of 18 1. Introduction Surfactants are amphiphilic molecules with a hydrophilic and a hydrophobic group, able to reduce surface tension. These molecules are largely used in the environmental, food, pharmaceutical, medical, and cleaning industries, among others [ 1 – 3 ]. Biosurfactants are secondary metabolites produced by bacteria, yeast, or fungi, capable of reducing the surface tension of extracellular media [ 4 ]. They can have a variety of structures, mainly divided into six major groups, namely, glycolipids, lipopolysaccharides, lipopeptides and phospholipids, and hydroxylated and cross-linked fatty acids [ 5 ]. When compared with synthetic surfactants, biosurfactants have higher biodegradability, lower toxicity, and higher activity at extreme conditions, such as high temperatures, pH, and salinity [6]. Bacterial species of the genus Serratia belonging to the family Enterobacteriaceae are Gram-negative, facultative anaerobic, and rod-shaped bacteria [ 7 , 8 ]. They have been isolated from different environments, such as water, soil, plants, insects, and vertebrates [ 9 ]. Surfaces in these habitats have a variety of characteristics and can be hydrophobic, hydrophilic, fractal, smooth, and axenic, among others. To colonize them, bacteria from the genus Serratia produce biosurfactants (extracellular lipopeptides) as the serratomolides serrawettin W1 [10], serrawettin W2 [4], and serrawettin W3 [11]. Mutants for the serratomolides biosynthetic genes failed to form colonies on nutritive agar plates and these lipopeptides were suggested as promoters of a new type of spreading growth [4]. Both the serrawettin W1 and W2 biosynthetic genes (swrW and swrA genes) belong to a Non-Ribosomal Peptide Synthetase (NRPS) gene cluster [ 12 , 13 ]. Condensation, adenylation, thiolation, and thioesterase domains are detectable in both serrawettin gene clusters. Along with other proteins, such as PPTase or acyl carrier proteins (ACP) [ 12 , 14 ], serrawettin W1 and W2 biosynthetic proteins are able to synthesize serrawettin W1 and W2 lipopeptides. Serrawettin W1 has a symmetric structure composed of two serine amino acids and two fatty acids (3-hydroxydecanoic) [ 4 ] while serrawettin W2 is a cyclic peptide containing five amino acids. This lipopeptide promotes flagellum spreading growth, contributing specifically to surface bacterial translocation [ 4 ], and was able to demonstrate antimicrobial activity against many bacteria and fungi and an antitumoral activity against Hela cells [ 13 ]. The Serratia strains producing serrawettin W1 showed the highest broad-spectrum antimicrobial activity against clinical, food, and environmental bacterial pathogens compared with the Serratia strains producing serrawettin W2 [ 15 ]. A strain of S. marcescens showed the capacity to inhibit an endophytic fungus due to its production of compounds, namely, serrawettins [16]. Due to the limited information on this topic, in this study, we performed a comparative genomic analysis to determine the distribution and the organization of the serrawettins biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. Here, we report a deep analysis of the serrawettin W1 and W2 gene clusters and establish their organization using antiSMASH software. In particular, for the first time, we show the serrawettin W1 gene clusters’ organization. These analyses showed the presence of four genes common to all the serrawettin gene clusters, highlighting their key potential in the serrawettins biosynthetic process. 2. Materials and Methods 2.1. Bacterial Strains, Data Collection, and Genomes Eighty-four Serratia genomes available on NCBI, representing all the publicly available genomes, were included in this study (Table S1), obtained from 49 different hosts and isolated from 29 countries [13,17–49]. Serrawettin W1 and W2 biosynthetic gene cluster prediction was performed in all the 84 complete and draft Serratia genomes publicly available, using the web platform antiSMASH 3.0 [ 50 ]. This input dataset includes strains belonging to S. marcescens,S. liquefaciens,S. grimesii,S. nematodiphila, S. plymuthica , and S. ureilytica, as well as strains not characterized to the species level. AntiSMASH analysis output was examined to identify the serrawettins biosynthetic genes clusters. Therefore, to find the swrW and swrA genes, BLAST analysis was performed for all protein sequences, codified by Biology 2020,9, 482 3 of 18 each gene of the NRPS predicting metabolite clusters, using the NCBI [ 51 ] database. Cluster boundaries were predicted with the CASSIS algorithm specified for the NRPS domains [50]. 2.2. Phylogenetic Analysis The 16S rRNA sequences of the 47 Serratia strains, which showed the presence of serrawettins genes in their genomes (Table 1), were selected for phylogenetic analysis. These sequences were compared with the sequences available in the EMBL/GenBank database using the BLASTN network services [ 51 ], and with sequences available at the Eztaxon-e server (http://eztaxon-e.ezbiocloud.net/) [ 52 ]. Sequences were aligned within the SINA alignment service [ 53 ]. Sequences were included in the 16S rRNA-based Living Tree Project (LTP, release 115) database (http://www.arb-silva.de/projects/living-tree/) by parsimony implemented in the ARB software package version 5.5 [ 54 ]. Phylogenetic dendrograms of this study strains and closest reference sequences were constructed using the Neighbor-Joining and Randomized Axelerated Maximum Likelihood (RAxML) method with the GTRGAMMA model [ 55 ] included in the ARB software [54]. Table 1. Serratia strains with the swrW and swrA genes identified through antiSMASH software. AntiSMASH software was used in 84 Serratia genomes (see Table S1). X represents the presence of genes swrW and swrA in seventeen and eleven genomes, respectively. Bacterial Strain Accession Number swrW swrA Serratia sp. AS12 CP002774.1 X Serratia sp. AS13 CP002775.1 X Serratia sp. FS14 CP005927.1 X Serratia sp. SCBI CP003424.1 X Serratia sp. YD25 CP016948.1 X Serratia sp. SSNIH1 CP026383.1 X Serratia sp. PWN146 LT575490.1 X Serratia marcescens strain UMH8 CP018927.1 X Serratia marcescens strain IOMTU 115 AB894481.1 X Serratia marcescens subsp. marcescens ATCC 13880 JMPQ01000033.1 X Serratia marcescens strain CDC_813-60 DP21 JOVM01000004.1 X Serratia nematodiphila DZ0503SBS1 strain DSM 21420 JPUX00000000.1 X Serratia marcescens VGH107 AORJ00000000.1 X Serratia marcescens EGD-HP20 AVSR00000000.1 X Serratia marcescens WW4 CP003959.1 X Serratia marcescens BIDMC 81 JJMZ01000006.1 X Serratia strain TEL NODE_13 LDEG01000018.1 X Serratia plymuthica NBRC 102599TBCTU00000000.1 X Serratia grimesii isolate BXF1 LT883155.1 X Serratia grimesii strain A2 JGVP00000000.1 X Serratia plymuthica AS9 CP002773.1 X Serratia marcescens SM39 AP013063.1 X Serratia marcescens SmUNAM836 CP012685.1 X Serratia marcescens BIDMC 44 JAPD01000005.1 X Serratia ureilytica Lr5/4 LG59 JSFB01000001.1 X Serratia marcescens RSC-14 CP012639.1 X Serratia marcescens subsp. marcescens AH0650_Sm1 AG2 LFJS01000014.1 X Serratia marcescens subsp. marcescens Db11 HG326223.1 X 2.3. Serrawettins Biosynthetic Gene Clusters Analysis The NRPS gene cluster, where both the serrawettin W1 and W2 biosynthetic genes belong, was analyzed here by using antiSMASH software [ 50 ]. The reconstruction of the domains, modules, and structures of the serrawettins was performed by using Phyre2 tridimensional prediction [ 56 ], using the serrawettin W1 biosynthetic protein from Serratia sp. AS13, and W2 from Serratia sp. PWN146. PubChem 2D was used to explore the chemical information of the serrawettins using the bacterial models mentioned above. The concatenated amino acid sequences of each serrawettin biosynthetic gene cluster were organized by protein identification in the same order to allow a cluster alignment with ClustalW Biology 2020,9, 482 4 of 18 in MEGAX software [ 57 ]. The serrawettin W1 biosynthetic proteins from 17 Serratia genomes and serrawettin W2 biosynthetic proteins from 11 Serratia genomes were separately aligned. The evolutionary relationship between the clusters and between the serrawettin biosynthetic protein sequences were established using the Neighbor-Joining method, Poisson model [ 58 ], in MEGAX software [ 57 ]. The aligned and organized cluster genes were represented in an evolutionary tree according to the size, direction, and accession numbers identified with the NCBI BLAST database [ 51 ]. Functions of the core proteins in all clusters were searched on UniProt [59]. 3. Results 3.1. Bacterial Phylogeny and Comparative Genomics of Serratia spp. All the 84 Serratia strains selected for this study had their genome publicly available at NCBI. According to the Neighbor-Joining and Maximum-Likelihood phylogenetic trees based on 16S rRNA gene sequences, 14 strains belong to S. marcescens, two strains belong to S. plymuthica, two strains belong to S. grimesii, one strain belongs to S. liquefaciens, one stain belongs to S. nematodiphila, one strain belongs to S. ureilytica, and 63 strains could not be assigned to species level due to a similarity lower than 97% (Figure 1). Genomes of the Serratia strains have a size from 5.0 Mbp to 7.7 Mbp and the G +C content varies from 45.9 to 60.1 mol%. Biology 2020, 9, x 4 of 19 PubChem 2D was used to explore the chemical information of the serrawettins using the bacterial models mentioned above. The concatenated amino acid sequences of each serrawettin biosynthetic gene cluster were organized by protein identification in the same order to allow a cluster alignment with ClustalW in MEGAX software [57]. The serrawettin W1 biosynthetic proteins from 17 Serratia genomes and serrawettin W2 biosynthetic proteins from 11 Serratia genomes were separately aligned. The evolutionary relationship between the clusters and between the serrawettin biosynthetic protein sequences were established using the Neighbor-Joining method, Poisson model [58], in MEGAX software [57]. The aligned and organized cluster genes were represented in an evolutionary tree according to the size, direction, and accession numbers identified with the NCBI BLAST database [51]. Functions of the core proteins in all clusters were searched on UniProt [59]. 3. Results 3.1. Bacterial Phylogeny and Comparative Genomics of Serratia spp. All the 84 Serratia strains selected for this study had their genome publicly available at NCBI. According to the Neighbor-Joining and Maximum-Likelihood phylogenetic trees based on 16S rRNA gene sequences, 14 strains belong to S. marcescens, two strains belong to S. plymuthica, two strains belong to S. grimesii, one strain belongs to S. liquefaciens, one stain belongs to S. nematodiphila, one strain belongs to S. ureilytica, and 63 strains could not be assigned to species level due to a similarity lower than 97% (Figure 1). Genomes of the Serratia strains have a size from 5.0 Mbp to 7.7 Mbp and the G + C content varies from 45.9 to 60.1 mol%. Figure 1. A phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequence of the Serratia strains used in this study and the type of strains. The tree was created using the NeighborJoining method in ARB software. The numbers on the tree indicate the percentages of bootstrap sampling, derived from 1000 replications; values below 50% are not shown. In blue are shown the Serratia strains that showed the presence of the serrawettin W1 biosynthetic gene cluster and in green are shown the Serratia strains that showed the presence of the serrawettin W2 biosynthetic gene cluster. The type species Escherichia coli DSM 30083T was used as the outgroup. Scale bar, 1 inferred nucleotide substitution per 100 nucleotides. Figure 1. A phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequence of the Serratia strains used in this study and the type of strains. The tree was created using the Neighbor-Joining method in ARB software. The numbers on the tree indicate the percentages of bootstrap sampling, derived from 1000 replications; values below 50% are not shown. In blue are shown the Serratia strains that showed the presence of the serrawettin W1 biosynthetic gene cluster and in green are shown the Serratia strains that showed the presence of the serrawettin W2 biosynthetic gene cluster. The type species Escherichia coli DSM 30083 T was used as the outgroup. Scale bar, 1 inferred nucleotide substitution per 100 nucleotides. Bioinformatic analysis through antiSMASH software showed that the serrawettin W1 biosynthetic gene was present in 17 Serratia genomes, strains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599 T , BXF1, A2, AS13, AS9, and AS12 (Table 1and Table S2). The serrawettin W2 biosynthetic gene was present in 11 Biology 2020,9, 482 5 of 18 Serratia strains, PWN146, SSNIH1, SM39, SmUNAM836, BIDMC 44, Lr5/4 LG59, RSC-14, AH0650_Sm1 AG2, Db11, SCBI, and YD25T(Table 1and Table S3). 3.2. Serrawettin W1 Biosynthetic Gene Clusters All serrawettin W1 biosynthetic gene clusters were identified as NRPS clusters. Moreover, the bioinformatic analysis predicted an architecture including the domains condensation (C), adenylation (A), thiolation (T), and thioesterase (TE) in all the serrawettin W1 biosynthetic genes (Figure 2). Biology 2020, 9, x 5 of 19 Bioinformatic analysis through antiSMASH software showed that the serrawettin W1 biosynthetic gene was present in 17 Serratia genomes, strains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599T, BXF1, A2, AS13, AS9, and AS12 (Tables 1 and S2). The serrawettin W2 biosynthetic gene was present in 11 Serratia strains, PWN146, SSNIH1, SM39, SmUNAM836, BIDMC 44, Lr5/4 LG59, RSC14, AH0650_Sm1 AG2, Db11, SCBI, and YD25T (Tables 1 and S3). 3.2. Serrawettin W1 Biosynthetic Gene Clusters All serrawettin W1 biosynthetic gene clusters were identified as NRPS clusters. Moreover, the bioinformatic analysis predicted an architecture including the domains condensation (C), adenylation (A), thiolation (T), and thioesterase (TE) in all the serrawettin W1 biosynthetic genes (Figure 2). Figure 2. Serrawettin W1: from the biosynthetic gene cluster to the structure of serrawettin W1. Genetic organization of the genome of Serratia strain AS13 by antiSMASH analysis, Phyre2 tridimensional prediction of serrawettin W1 biosynthetic protein, and PubChem 2D structure of serrawettin W1. (a) Serrawettin W1 biosynthetic gene cluster with identification of the srwW core gene; (b) swrW gene organization: C, condensation domain; A, adenylation domain; T, thiolation domain; and TE, thioesterase domain; (c) biosynthetic protein with the condensation, adenylation, thiolation, and thioesterase domains; (d) serrawettin W1 2D structure. To confirm the identification of the swrW gene revealed by antiSMASH, each swrW was queried to NCBI BLASTP, in order to find the closest relative and determine the identity percentage (Tables 2 and S2). The protein from the serrawettin W1 biosynthetic gene (swrW) showed an identity percentage that ranges from 77.79% to 100% as the closest identification by using BLASTP (Table 2). Figure 2. Serrawettin W1: from the biosynthetic gene cluster to the structure of serrawettin W1. Genetic organization of the genome of Serratia strain AS13 by antiSMASH analysis, Phyre2 tridimensional prediction of serrawettin W1 biosynthetic protein, and PubChem 2D structure of serrawettin W1. ( a ) Serrawettin W1 biosynthetic gene cluster with identification of the srwW core gene; ( b )swrW gene organization: C, condensation domain; A, adenylation domain; T, thiolation domain; and TE, thioesterase domain; ( c ) biosynthetic protein with the condensation, adenylation, thiolation, and thioesterase domains; (d) serrawettin W1 2D structure. To confirm the identification of the swrW gene revealed by antiSMASH, each swrW was queried to NCBI BLASTP, in order to find the closest relative and determine the identity percentage (Table 2 and Table S2). The protein from the serrawettin W1 biosynthetic gene (swrW) showed an identity percentage that ranges from 77.79% to 100% as the closest identification by using BLASTP (Table 2). Biology 2020,9, 482 6 of 18 Table 2. The serrawettin W1 gene (swrW) of each strain in this study and the accession numbers and identification of the swrW gene’s closest relatives using BLASTP. Bacterial Strain swrW Closest Relative Genes Accession Number Identity Percentage Serratia marcescens EGD-HP20 non-ribosomal peptide synthetase ERH70695.1 99.52 Serratia marcescens WW4 serrawettin W1 synthetase AGE20181.1 100 Serratia sp. FS14 putative serrawettin W1 synthetase AIA46701.1 100 Serratia marcescens VGH107 amino acid adenylation protein EMF04443.1 99.12 Serratia nematodiphila DZ0503SBS1 strain DSM 21420 non-ribosomal peptide synthetase KFF87803.1 99.31 Serratia marcescens strain IOMTU 115 putative serrawettin W1 synthetase BAO21138.1 99.92 Serratia marcescens strain UMH8 non-ribosomal peptide synthetase ASM18665.1 99.01 Serratia marcescens subsp. marcescens ATCC 13880 amino acid adenylation domain-containing protein KFD14984.1 98.57 Serratia marcescens strain CDC_813-60 DP21 non-ribosomal peptide synthetase KFL05097.1 98.63 Serratia marcescens BIDMC 81 non-ribosomal peptide synthetase EZQ62923.1 95.12 Serratia strain TEL NODE_13 non-ribosomal peptide synthetase KLE36484.1 95.05 Serratia plymuthica NBRC 102599T non-ribosomal peptide synthetase WP_063202307.1 81.71 Serratia sp. AS13 non-ribosomal peptide synthetase AEG30284.1 81.48 Serratia plymuthica AS9 non-ribosomal peptide synthetase AEF47625.1 81.48 Serratia sp. AS12 non-ribosomal peptide synthetase WP_013814722.1 81.48 Serratia grimesii isolate BXF1 amino acid adenylation domain-containing protein SMZ58711.1 77.79 Serratia grimesii strain A2 non-ribosomal peptide synthetase KFB89923.1 78.17 Eighty different genes from the serrawettin W1 biosynthetic gene cluster were identified. Fifteen genes are common to all 17 gene clusters (Figure 3and Table S4), such as genes encoding for murein hydrolase effector protein LrgB and murein hydrolase regulator LrgA, both with hydrolase activity; LysR regulatory protein with DNA-binding transcription factor activity; a sodium-hydrogen antiporter and xanthine-uracil-vitamin C permease, both with transmembrane transport activity; glyoxalase–bleomycin resistance protein and glutathione S-transferase domain protein, both with a dioxygenase activity; 3-oxoacyl-(acyl-carrier-protein) reductase; single-stranded DNA-binding protein; exonuclease ABC subunit A; maltose O-acetyltransferase; and aromatic amino acid aminotransferase. Not considering strain NRBC 102599 T , an additional seven genes were found to be common to all strains in the serrawettin W1 biosynthetic gene cluster (Figure 3). Moreover, 15 genes are exclusive to the S. plymuthica NBRC 102599Tbiosynthetic gene cluster (Figure 3). Biology 2020,9, 482 7 of 18 Biology 2020, 9, x 7 of 19 Figure 3. Serrawettin W1 gene cluster analysis. The phylogenetic relationship was established with Mega X software by the Neighbor-Joining method on aligned serrawettin W1 gene clusters of Serratia strains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599, BXF1, A2, AS13, AS9, and AS12. (a) Phylogenetic tree based on protein sequences of the serrawettin W1 biosynthetic gene cluster on an established genetic organization. (b) Serrawettin W1 biosynthetic gene clusters based on natural genetic organization. (c) Comparison of the phylogenetic tree based on protein sequences of the serrawettin W1 biosynthetic gene clusters (left) with the phylogenetic tree of the serrawettin W1 biosynthesis protein (right). The scale bar of 0.050 infers the nucleotide substitutions per 100 nucleotides. Figure 3. Serrawettin W1 gene cluster analysis. The phylogenetic relationship was established with Mega X software by the Neighbor-Joining method on aligned serrawettin W1 gene clusters of Serratia strains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599, BXF1, A2, AS13, AS9, and AS12. ( a ) Phylogenetic tree based on protein sequences of the serrawettin W1 biosynthetic gene cluster on an established genetic organization. ( b ) Serrawettin W1 biosynthetic gene clusters based on natural genetic organization. ( c ) Comparison of the phylogenetic tree based on protein sequences of the serrawettin W1 biosynthetic gene clusters (left) with the phylogenetic tree of the serrawettin W1 biosynthesis protein (right). The scale bar of 0.050 infers the nucleotide substitutions per 100 nucleotides. Biology 2020,9, 482 8 of 18 The relationship between strains established based on the analysis of the concatenated genes of the W1 biosynthetic gene cluster defined the same clusters as the relationships defined based on the swrW gene analysis, except for S. plymuthica NBRC 102599 T , which is discordant. The position of S. plymuthica NBRC 102599 T in W1 phylogenetic tree highlights the different gene composition of the W1 biosynthetic cluster of the strain. On the other hand, in the swrW phylogenetic tree, S. plymuthica NBRC 102599Tforms a sister group with Serratia strains AS13, AS9, and AS12 (Figure 3). In the serrawettin W1 biosynthetic gene cluster, software prediction identified four genes involved in PKS-NRPS (PolyKetide Synthases Non-Ribosomal Peptide Synthetases), encoding for enoylreductase quinone oxidoreductase (only present in seven strains), ketoreductase 3-oxoacyl-(acyl-carrier-protein) reductase (present in all strains), enoylreductase dehydrogenase (absent in two strains), and aromatic amino acid aminotransferase (present in all strains) (Table 3and Table S4, Figure 3). Table 3. The PKS gene accession numbers from the swrW biosynthetic gene clusters predicted by antiSMASH software. Bacterial Strain Enoylreductase Quinone Oxidoreductase Ketoreductase 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase Enoylreductase Dehydrogenase Aromatic Amino Acid Aminotransferase Serratia marcescens EGD-HP20 ERH70706.1 ERH70710.1 ERH70714.1 Serratia marcescens WW4 AGE20192.1 AGE20197.1 AGE20201.1 Serratia sp. FS14 AIA46690.1 AIA46685.1 AIA46681.1 Serratia marcescens VGH107 EMF04432.1 EMF04427.1 EMF04423.1 Serratia nematodiphila DZ0503SBS1 strain DSM 21420 KFF87792.1 KFF87787.1 KFF87783.1 Serratia marcescens strain IOMTU 115 BAO21148.1 BAO21153.1 BAO21155.1 Serratia marcescens strain UMH8 ASM18675.1 ASM18680.1 ASM18684.1 Serratia marcescens subsp. Marcescens ATCC 13880 KFD14974.1 KFD14969.1 KFD14965.1 Serratia marcescens strain CDC_813-60 DP21 KFL04091.1 KFL03204.1 KFL04717.1 Serratia marcescens BIDMC 81 EZQ62938.1 EZQ62913.1 EZQ62903.1 Serratia strain TEL NODE_13 KLE36470.1 KLE36494.1 KLE36503.1 Serratia plymuthica NBRC 102599TWP_063202297.1 WP_062868864.1 WP_006328339.1 Serratia sp. AS13 AEG30270.1 AEG30294.1 AEG30297.1 AEG30301.1 Serratia plymuthica AS9 AEF47611.1 AEF47635.1 AEF47638.1 AEF47642.1 Serratia sp. AS12 WP_013814712.1 WP_013814732.1 WP_013814734.1 WP_013814736.1 Serratia grimesii isolate BXF1 SMZ58698.1 SMZ58721.1 SMZ58727.1 SMZ58731.1 Serratia grimesii strain A2 KFB89936.1 KFB89913.1 KFB89904.1 3.3. Serrawettin W2 Biosynthetic Gene Clusters Every serrawettin W2 biosynthetic gene (swrA) showed an architecture composed of five modules, each with a condensation (C1, C2, C3, C4, and C5), adenylation (A1, A2, A3, A4, and A5), and thiolation (T1, T2, T3, T4, and T5) domain. Module 5 has an additional thioesterase (TE) domain. This organization is shared by all the serrawettin W2 biosynthetic genes (Figure 4). Biology 2020,9, 482 9 of 18 Biology 2020, 9, x 9 of 19 Figure 4. Serrawettin W2: from the biosynthetic gene cluster to the structure of serrawettin W2. Genetic organization of the genome of Serratia strain PWN146 by antiSMASH analysis, Phyre2 tridimensional prediction of the serrawettin W2 biosynthetic protein, and PubChem 2D structure of serrawettin W2. (a) Serrawettin W2 biosynthetic gene cluster with identification of the swrA core gene; (b) swrA gene organization: five modules (M1–M5) with C, condensation domain; A, adenylation domain; T, thiolation domain; and TE, thioesterase domain; (c) biosynthetic protein with five modules (M1–M5), each composed of condensation, adenylation, and thiolation domains, and in the last module an additional thioesterase domain. To confirm the identification of the swrA gene revealed by antiSMASH, each swrA was, as mentioned above, queried to NCBI BLASTX, in order to find the closest relative and determine the identity percentage (Tables 4 and S3). The protein coded by the biosynthetic genes of serrawettin W2 (swrA) showed an identity percentage that ranges from 76.38% to 99.4% as the closest identification by using BLASTX (Table 4). Table 4. The serrawettin W2 gene (swrA) of each strain in this study and the accession numbers and identification of the swrA gene’s closest relatives using BLASTX. Bacterial Strain swrA Closest Relative Genes Accession Number Identity Percentage Serratia marcescens SM39 Serratia marcescens SM39 DNA, complete genome BAO35825.1 76.70 Serratia marcescens SmUNAM836 Serratia marcescens strain SmUNAM836, complete genome ALE98111.1 76.70 Serratia sp. SSNIH1 Serratia sp. SSNIH1 chromosome, complete genome AUY16873.1 76.70 Serratia sp. PWN146 Serratia marcescens isolate PWN146_assembly genome assembly, chromosome SAY45247.1 77.02 Serratia ureilytica Lr5/4 LG59 Serratia marcescens strain UMH7, complete genome KKO55915.1 76.38 Serratia marcescens RSC-14 Serratia marcescens strain RSC-14, complete genome ALD45109.1 76.38 Serratia marcescens BIDMC 44 Serratia marcescens strain 1274 genome ETX44761.1 77.35 Serratia sp. YD25 Serratia sp. YD25, complete genome AOF02338.1 93.57 Serratia sp. SCBI Serratia sp. SCBI, complete genome AIM23801.1 93.44 Serratia marcescens subsp. marcescens AH0650_Sm1 AG2 Serratia marcescens strain BWH-23 chromosome, complete genome KMU50701.1 99.40 Serratia marcescens subsp. marcescens Db11 Serratia marcescens subsp. marcescens Db11, complete genome CDG14244.1 99.32 Figure 4. Serrawettin W2: from the biosynthetic gene cluster to the structure of serrawettin W2. Genetic organization of the genome of Serratia strain PWN146 by antiSMASH analysis, Phyre2 tridimensional prediction of the serrawettin W2 biosynthetic protein, and PubChem 2D structure of serrawettin W2. ( a ) Serrawettin W2 biosynthetic gene cluster with identification of the swrA core gene; ( b )swrA gene organization: five modules (M1–M5) with C, condensation domain; A, adenylation domain; T, thiolation domain; and TE, thioesterase domain; ( c ) biosynthetic protein with five modules (M1–M5), each composed of condensation, adenylation, and thiolation domains, and in the last module an additional thioesterase domain. To confirm the identification of the swrA gene revealed by antiSMASH, each swrA was, as mentioned above, queried to NCBI BLASTX, in order to find the closest relative and determine the identity percentage (Table 4and Table S3). The protein coded by the biosynthetic genes of serrawettin W2 (swrA) showed an identity percentage that ranges from 76.38% to 99.4% as the closest identification by using BLASTX (Table 4). Table 4. The serrawettin W2 gene (swrA) of each strain in this study and the accession numbers and identification of the swrA gene’s closest relatives using BLASTX. Bacterial Strain swrA Closest Relative Genes Accession Number Identity Percentage Serratia marcescens SM39 Serratia marcescens SM39 DNA, complete genome BAO35825.1 76.70 Serratia marcescens SmUNAM836 Serratia marcescens strain SmUNAM836, complete genome ALE98111.1 76.70 Serratia sp. SSNIH1 Serratia sp. SSNIH1 chromosome, complete genome AUY16873.1 76.70 Serratia sp. PWN146 Serratia marcescens isolate PWN146_assembly genome assembly, chromosome SAY45247.1 77.02 Serratia ureilytica Lr5/4 LG59 Serratia marcescens strain UMH7, complete genome KKO55915.1 76.38 Serratia marcescens RSC-14 Serratia marcescens strain RSC-14, complete genome ALD45109.1 76.38 Serratia marcescens BIDMC 44 Serratia marcescens strain 1274 genome ETX44761.1 77.35 Serratia sp. YD25 Serratia sp. YD25, complete genome AOF02338.1 93.57 Serratia sp. SCBI Serratia sp. SCBI, complete genome AIM23801.1 93.44 Serratia marcescens subsp. marcescens AH0650_Sm1 AG2 Serratia marcescens strain BWH-23 chromosome, complete genome KMU50701.1 99.40 Serratia marcescens subsp. marcescens Db11 Serratia marcescens subsp. marcescens Db11, complete genome CDG14244.1 99.32 Sixty-eight genes were identified in the serrawettin W2 biosynthetic gene clusters (Figure 5and Table S5). Twenty-four genes were present in all the strains’ gene clusters (Figure 5), namely, the Biology 2020,9, 482 16 of 18 26. Abebe, E.; Abebe-Akele, F.; Morrison, J.; Cooper, V.; Thomas, W.K. An insect pathogenic symbiosis between aCaenorhabditis and Serratia.Virulence 2011,2, 158–161. [CrossRef] [PubMed] 27. Lephoto, T.E.; Featherston, J.; Gray, V.M. Draft Whole-genome sequence of Serratia sp. strain TEL, associated with Oscheius sp. TEL-2014 (Nematoda: Rhabditidae) isolated from a grassland in South Africa. Genome Announc. 2015,3, e00747-15. [CrossRef] [PubMed] 28. 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