Abstract
Pseudomonas aeruginosa and Sphinogobacterium sp. are well known for their ability to decontaminate many environmental pollutants like PAHs, dyes, pesticides and plastics. The present study reports the annotation of genomes from P. aeruginosa and Sphinogobacterium sp. that were isolated from compost, based on their ability to degrade poly(lactic acid), PLA, at mesophillic temperatures (~30°C). Draft genomes of both the strains were assembled from Illumina reads, annotated and viewed with an aim of gaining insight into the genetic elements involved in degradation of PLA. The draft-assembled genome of strain Sphinogobacterium strain S2 was 5,604,691 bp in length with 435 contigs (maximum length of 434,971 bp) and an average G+C content of 43.5%. The assembled genome of P. aeruginosa strain S3 was 6,631,638 bp long with 303 contigs (maximum contig length of 659,181 bp) and an average G+C content 66.17 %. A total of 5,385 (60% with annotation) and 6,437 (80% with annotation) protein-coding genes were predicted for strains S2 and S3 respectively. Catabolic genes for biodegradation of xenobiotic and aromatic compounds were identified on both draft genomes. Both strains were found to have the genes attributable to the establishment and regulation of biofilm, with more extensive annotation for this in S3. The genome of P. aeruginosa S3 had the complete cascade of genes involved in the transport and utilization of lactate while Sphinogobacterium strain S2 lacked lactate permease, consistent with its inability to grow on lactate. As a whole, our results reveal and predict the genetic elements providing both strains with the ability to degrade PLA at mesophilic temperature.
1. Introduction
Poly(lactic acid) (PLA), is a bio-based aliphatic polyester polymer, obtained from sources such as corn sugar, cassava, wheat, rice, potato, and sugar cane, considered renewable [1, 2]. PLA is completely biodegradable under industrial composting conditions [3] as well as under unsupervised environmental conditions where its biodegradation is considered safe [4]. In the last two decades biodegradation of PLA has been extensively studied and many microbial species (actinomycete, bacteria, fungus) have been identified with the ability to degrade PLA [4]. Most of the reported bacterial species are from the family Pseudonocardiaceae, Thermomonosporaceae, Micromonosporaceae, Streptosporangiaceae, Bacillaceae and Thermoactinomycetaceae while the fungal species are mainly from the phylum Basidiomycota (Tremellaceae) and Ascomycota (Trichocomaceae, Hypocreaceae) [5–11].
In our previous study we also described four bacterial strains designated as S1, S2, S3 and S4, able to degrade PLA at ambient temperature [12]. Two of the isolated strains, Sphingobacterium sp. (S2) and P. aeruginosa (S3), were also evaluated for their PLA degradation in soil microcosms [13]. The genus Sphingobacterium is from Phylum Bacteriodetes, Family Sphingobacteriace, named with reference to the sphingolipids in their cell wall [14, 15]. They are gram-negative rods and the GC content of their DNA is usually ranging from 35 to 44 mol% [16, 17]. Sphingobacterium sp. are found in a range of habitats like soil, forest, compost, activated sludge, rhizosphere, faeces, lakes and various food sources [18]. Sphingobacterium had also been reported to have their potential role in biodegradation of different pollutants including mixed plastic waste, PAHs, biodegradation of oil and pesticides [19–21]. Pseudomonas aeruginosa are gram-negative bacteria from γ-subdivision of proteobacteria. They are ubiquitously distributed in soil and aquatic habitats and are well-known opportunistic pathogens [22, 23]. It has the ability to thrive in highly diverse and unusual ecological niches with scarce available nutrients. Its metabolic versatility allows it to survive on a variety of diverse carbon sources for its survival, even in some disinfectants. and can metabolize many antibiotics [24, 25]. In previous reports role of Pseudomonas aeruginosa in degradation of different polymers including PAHs, biodegradation of xenobiotic compounds, degradation of oil, dyes and plastics is well documented [26–30],
PLA degrading bacteria reported in our previous study [12] were isolated from compost and had the ability to degrade PLA at ambient temperature. Interestingly, our P. aeruginosa strains were lactate utilizing while the Sphingobacterium sp. and Chryseobacterium sp. strains were unable to utilize lactate when provided as the sole carbon source in minimal media. We also observed that all four isolates could form biofilm on PLA. The purpose of this study was to analyze the genomes of two of our isolates and explore the genetic determinants responsible for conferring the particular characteristics to the strains promoting their degradation ability. The genes controlling lactate utilization and biofilm formation and regulation were identified. Whole genome sequence analysis for P. aeruginosa is extensive but such data for Sphingobacterium sp. is quite limited. To our knowledge this is first report that gives such genetic depth to PLA degrading bacterial strains and will provide the basis for further analysis.
2. Material and methods
2.1 DNA extraction
Two of our previously isolated, PLA degrading bacterial strains, Sphingobacterium sp. strain S2 and P. aeruginosa strain S3 (GenBank accession numbers KY432687 and KY432688, respectively) were selected for genome sequencing [12]. Both of these strains were grown separately in 100 mL of LB in a 250 mL Erlenmeyer flask for 16 hours in a shaking incubator at 30°C and 70 rpm. Genomic DNA was subsequently isolated by using MO BIO PowerSoil® DNA isolation kit (MO BIO laboratories, Inc. Loker Ave west, Carlsbad, CA). NanoDrop® ND-1000 spectrophotometer and ND-1000 V3.1.8 software (Wilmington, DE, USA) was used to determine DNA concentrations of purified samples and sent for whole genome sequencing at Michigan State University Genomics Facility (MSU-RTSF).
2.2 Genome sequencing
Libraries for sequencing were prepared using the Illumina TruSeq Nano DNA Library Preparation Kit on a Perkin Elmer Sciclone NGS robot. Before sequencing, the qualities of the libraries were tested and quantification was performed using a combination of Qubit dsDNA HS, Caliper LabChip GX HS DNA and Kapa Illumina Library Quantification qPCR assays. Libraries were pooled in equimolar quantities and loaded on an Illumina MiSeq standard v2 flow cell with a 2×250bp paired end format and using a v2 500 cycle reagent cartridge. Illumina Real Time Analysis (v1.18.64) was used for base calling and the output was converted to FastQ format with Illumina Bc12fastq (v1.8.4) after demultiplexing. A total of 6,304,420 reads (~3.15 GB) were obtained for strain S2 and 5,800,229 reads (~2.9GB) were obtained for strain S3.
2.3 Sequence assembly, annotation and analysis
Assembly of the whole genome was performed using the full Spades assembly function within PATRIC (Pathosystems Resource Integration Center) (PATRIC 3.4.9) as implemented in the miseq assembly option. This assembly option incorporates BayesHammer algorithms followed by Spades, (Spades version 3.8.). Rast tool kit as implemented in PATRIC (PATRIC 3.4.9) was used for the annotation of contigs. The assembled contig file generated from this assembly was used as seed for the Comprehensive Genome Analysis function in PATRIC. The genomes were interrogated for the distribution of specific protein families (PGFams) using the protein family sorter tool on PATRIC. The genomes were compared to their closest reference genomes available on PATRIC to examine the strain-specific unique proteins as well as proteins common to the closest relative using the filter option in protein family sorter tool on PATRIC.
2.4 Average nucleotide identity (ANI) for species delineation
Isolates were further analyzed using a whole genome based Average nucleotide identity (ANI) method to delineate the genomes to their correctly. ANI values were calculated using MiSI (microbial species identifier) tool that is publicly available at Integrated Microbial Genomes (IMG) database [31]. The algorithm used in the original method proposed by Konstantinidis and Tiedje was modified and used to determine ANI between two genomes [32]. The average of the nucleotide identity of the orthologous genes of the pair of genomes was calculated and identified as bidirectional best hits (BBHs) using a similarity search tool, NSimScan (http://www.scidm.org/). The ANI of one genome to other genome is defined as the sum of the %-identity times the alignment length for all best bidirectional hits, divided by the sum of the lengths of the BBH genes. This pairwise calculation is performed in both directions. The strains used for comparison were complete genomes obtained from NCBI and are as follows. For the Sphingobacterium comparisons; S. thalpophilum DSM 11723 (Draft genome of 32 contigs), S. sp. G1-14, S. sp. B29, S. multivorum DSM 11691, S. lactis DSM 22361, S. wenxiniae DSM 22789, S. mizutaii DSM 11724 and S. sp. 21. For the Pseudomonas aeruginosa comparisons; P. aeruginosa PSE302, P. aeruginosa PA96, P. aeruginosa PA01H20, P. aeruginosa DSM 50071 P. aeruginosa PAO1, P. aeruginosa PAK, P. aeruginosa O12 PA7, P. aeruginosa PA_D25, P. aeruginosa PA_D1, P. aeruginosa KU and P. aeruginosa T52373.
2.5 Comparative alignments using MeDuSa and Mauve
For the comparative alignment of the genomes with their reference genomes and their visualization, MeDuSa [33] was used to reduce the number of contigs through comparison with the gene order of the closest strain. This was followed by alignment with MAUVE [34] to the same reference strain to provide an estimate of alignment similarity. P. aeruginosa PSE305 was used as a reference for our P. aeruginosa S3 while Sphingobacterium thalpophilum DSM 11723 was selected as a reference for Sphingobacterium sp. S2. References were selected based on closest ANI score.
3. Results and discussion
3.1. General Genome features of Sphingobacterium sp. (S2) and P. aeruginosa (S3)
The assembly of the draft genome for strain S2 yielded 87 contigs (434971 maximum length) and a total of 5,445,390 assembled base pairs with 43.66% G+C (Table 1). The largest contig was 434,944 bp. There were an estimated 4,951 CDS regions, 2,864 proteins with functional assignments and several antibiotic resistance genes. The assembled draft genome of S3 had 63 contigs (658,980 maximum length) with a total of 6,509,961 assembled base pairs and was 66.26% G+C. There were an estimated 6,239 CDS regions and 4,932 proteins with functional assignments and a substantial number of putative antibiotic resistant and virulence determinants.
The distribution of identified genes to cell subsystems is shown in Figure 1. Sphingobacterium S2 has more genes linked to protein processing, energy and membrane transport whereas P. aeruginosa S3 has more genes linked to metabolism, stress response/defense/virulence and cell envelop.
3.2 Genetic relatedness based on ANI
The average nucleotide identity (ANI) value describes the similarity between the sequences of the conserved regions of two genomes and measures the genetic relatedness between them [35]. ANI measurements are considered more informative over 16S rRNA gene identity as they are based on a larger number of genes [36]. ANI comparisons were done to explore the interspecies genetic relatedness between Sphingobacterium sp. strain S2 and P. aeruginosa strain S3 and other sequenced species from these genera. In the case of P. aeruginosa strain S3, Fig 1 shows a cluster was formed in the upper-right corner containing all the closely related strains with more than 98-99% gene identity based on 16S rRNA sequences and 93-97%. ANI. P. aeruginosa PSE305 showed highest ANI value and 16S rRNA gene identity of 97.69 % and 99 % respectively. Two or more strains with ANI values from 95% and above should be considered as the same species [37]. Accordingly, P. aeruginosa strain S3 and other strains included in the analysis belong to the same species. The results are consistent with by the phylogenetic results based on 16S rRNA gene identity as previously reported [12] but the ANI analysis indicated that our strain was closest to P. aeruginosa PSE305 rather than P. aeruginosa BUP2. Also, based on 16S rRNA comparative sequence analysis P. aeruginosa sv. O12 PA7 showed 99 % sequence similarity to P. aeruginosa strain S3 but had < 95 %. ANI value. ANI analysis for Sphingobacterium sp. strain S2 showed a different clustering of sequenced strains, some of which were not the same species. The data point in the upper-right corner represents Sphingbacterium thalpophilum DSM11723 which showed more than 98% 16S rRNA gene similarity and 98% ANI with Strain S2 and was the closest match to our strain. Two data points in the lower-right corner represented 97 and 98 % 16S rRNA gene identity but had ANI values of 79 and 80 % others having less than 95 % 16S rRNA gene identity had even lower ANI values. According to 16S rRNA gene identity and phylogenetic affiliations Sphingobacterium sp. strain S2 showed close resemblance to Sphingobacterium thalpophilum Y19 but ANI value showed Sphingbacterium thalpophilum DSM11723 to be the closest relative to our strain. Both of our Sphingobacterium sp. strain S2 and P. aeruginosa strain S3 were isolated from the compost samples, while the most closely related strains, Sphingbacterium thalpophilum DSM11723 and P. aeruginosa PSE305, were isolated from human clinical samples.
3.3 MAUVE and MEDUSA alignments
MAUVE and MEDUSA were used to find sequence homologies of the two isolates with the strains identified as closest in the database by ANI calculations. The alignments shown in Figure 3 were derived by first aligning the SPADE-assembled contigs against reference strains, (Sphingbacterium thalpophilum DSM11723 or P. aeruginosa PSE305) with MeDuSa (REF), followed by alignment in MAUVE using the MeDuSa ordered scaffold of isolates S2 & S3. While the closest Sphingobacterium genome (DSM11723) in the current IMG database was close, based on a 98.1% ANI, this was a draft sequence with 32 contigs (Fig. 3, Panel A). As a result, the MAUVE alignment shows large regions of homology but spotty consensus in terms of synteny. In contrast, our Pseudomonas strain was identified as P. aeruginosa with many finished genomes of this species in the IMG database. ANI analysis (above) indicated that P.aeruginosa strain PSE305 was the closest. This strain has a completed genome and as a result, serves as an excellent reference strain for our isolate. Panel B of Figure 3 shows the putative alignment of P.aeruginosa S3 with P. aeriginosa PSE305. The 63 contigs of the SPADE assembled strain S3 was reduced to 9 contigs after alignment against PSE305 with MeDuSa.
3.4 Metabolism
P. aeruginosa is a Gram-negative bacterium able to grow aerobically and anaerobically, when nitrate is available as terminal electron acceptor. It is capable of thriving in highly diverse and unusual ecological niches with low availability of nutrients. Its metabolic versatility allows it to use a variety of diverse carbon sources including certain disinfectants [38]. Moreover, it can synthesize a number of antimicrobial compounds [24, 25]. Using genome annotation through PATRIC, 1,084 ORFs in P.aeruginosa were assigned to metabolic pathways. Among the major pathways, 361 genes were assigned to amino acid metabolism had 361 genes assigned to it and 192, 286, 225 and 168 genes were assigned for energy metabolism, carbohydrate metabolism, metabolism of cofactors and vitamins and lipid metabolism, respectively. Metabolism of xenobiotics was allocated to 165 genes while 129 and 118 genes were nucleotide metabolism and biosynthesis of secondary metabolites had 129 and 118 genes assigned to their functions.
Sphingobacterium spp. are gram-negative rods, aerobic, exhibiting sliding motility and form yellow-pigmented colonies. Sphingobacteria have been isolated from diverse environments like soil, water, compost, deserts, blood and urine samples from human patients. A distinctive feature of Sphingobacterum is the presence of sphingolipids in their cell wall in high concentrations [14, 39]. In Sphingobacteria S2, 820 ORFs were assigned metabolic functions. 212 genes were identified as participating in amino acid metabolism while 262 genes were dedicated to carbohydrate metabolism. Energy metabolism and lipid metabolism had 148 and 132 genes identified, respectively, while 67 genes were assigned to xenobiotic biodegradation and metabolism. The disparity in assigned genes between Sphingobacterium and P. aeruginosa, particularly with respect to carbohydrate metabolism and amino acid synthesis is presented in Figure 4.
3.3. Xenobiotics biodegradation metabolism
In previous reports the role of P. aeruginosa in degradation of different polymers including PAHs, biodegradation of xenobiotic compounds, degradation of oil, dyes and plastics as well is well documented [26–30], Sphingobacterium had also been reported to have their potential role in biodegradation of different pollutants including mixed plastic waste, PAHs, biodegradation of oil and pesticides [19–21]. Table 2 describes the major pathways and the number of genes related to biodegradation of different xenobiotic compounds in both strains S2 and S3. According to sequence analysis, several pathways for the biodegradation of xenobiotic compounds were found in both strains with twice as many detected in P. aeruginosa as compared to Sphingobacterium sp. This may be due to the fact that P. aeruginosa is more comprehensively studied compared to Sphingobacterium sp. Among all the degradation pathways found in both strains, the degradation of benzoate in P. aeruginosa had the greatest number of annotated genes Benzoate, an aromatic compound, has been widely used as a model for the study of the bacterial catabolism of aromatic compounds. In Sphingobacterium sp. genes associated with 1,4-Dichlorobenzene degradation were detected and this compound is a well-studied halogenated aromatic hydrocarbon [40]. Many genes related to the degradation pathway of one of the most important class of pollutants, polycyclic aromatic hydrocarbons (PAHs) like naphthalene, anthracene, 1- and 2-methylnaphthalene, was found in both strains. Among the halogenated organic compounds, 29 genes were dedicated to tetrachloroethene degradation in P. aeruginosa S3, while 6 genes were found in Sphingobacterium sp. S2. For aromatic compounds and chlorinated aromatic compounds, pathways for the biodegradation of toluene, trinitrotoluene, xylene degradation, 1,4-Dichlorobenzene degradation and 2,4-Dichlorobenzoate were also found. Genes for the biodegradation of Bisphenol A, one of the most abundantly produced chemicals released into the environment and is a serious environmental pollutant, was also found in both of our strains. P. aeruginosa S3 was found to have 13 genes for bisphenol A degradation pathway[41]. Pathways for pesticides degradation like 1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) degradation and atrazine biodegradation were also found in both the isolates.
3.4. Lactate metabolism
Lactate utilization as sole carbon source is a property of many bacteria, where a key step of the process is oxidation of lactate [42–46]. Lactate dehydrogenases found in microbes are of two types, NAD-dependent lactate dehydrogenases (nLDHs) and NAD-independent lactate dehydrogenases (iLDHs), also called respiratory lactate. The latter is usually considered to be the enzyme mainly responsible for metabolism of lactate as a carbon source [45]. The lactate utilization system is comprised of three main membrane bound proteins: NAD-independent L-lactate dehydrogenase (L-iLDH), NAD-independent D-lactate dehydrogenase (D-iLDH), and a lactate permease (LldP). Lactate permease, LldP is responsible to take up lactate into the cells and lactate dehydrogenases carry out the oxidation of either form of lactate to pyruvate [47, 48]. In pathogenesis of some microbes role of lactate utilization has been observed [46]. Lactate utilization in pathogens not only has stimulatory effect on their growth, it also enhances synthesis of pathogenic determinants and imparts them resistance against various bactericidal mechanisms [46]. Utilization of lactate by different Pseudomonas strains is very well documented [44, 49–51]. In sequence analysis of our P. aeruginosa strain S3, a complete cascade of genes was found, encoding the machinery for lactate utilization that included a L-lactate permease, both L-lactate dehydrogenase and D-lactate dehydrogenase and a Lactate-responsive regulator LldR Table 3. This strain was isolated and characterized for its potential to degrade Poly(lactic acid), one of the more promising of the bio-based and biodegradable polymers currently in the market, and its potential to utilize lactate, one of the final products of PLA degradation, as a sole carbon source was already established in our previous study [12]. Presence of the lactate utilization machinery found through genome sequencing is the confirmation of our previous findings regarding lactate utilization by P. aeruginosa, strain S3. It was previously described in the literature that both L-iLDH and D-iLDH are present in the single operon and are induced coordinately in all the reported Pseudomonas strains. Expression of both enzymes is controlled by the presence of enantiomer of lactate [48]. In previous studies, it was reported that in P. aeruginosa strain XMG lactate utilization operon lldPDE consists of genes for lactate permease LldP, the L-lactate dehydrogenase LldD and the D-lactate dehydrogenase LldE and a nearby located lldR gene, which codes for a regulator LldR [48, 52]. In the genome analysis of our isolate Sphingobacterium strain S2, we found an incomplete set of genes, both L-lactate dehydrogenase and D-lactate dehydrogenase were present but no lactate permease was detected Table 3. Absence of lactate permease suggested that the strain is incapable of utilizing lactic acid as carbon source. This again confirmed our previous findings that showed that Sphingobacterium strain S2 did not utilize lactic acid as sole source of carbon. Sphingobacterium strain S2 was isolated and characterized based on its ability to degrade PLA suggesting that another degradation product of PLA was utilized for growth. Inability to grow on lactic acid had been previously reported in literature for different strains of Sphingobacterium [18, 53].
3.5. Genetic determinants for biofilm formation and regulation
In contrast to the planktonic life style, cells within a biofilm matrix are in close proximity where secreted enzymes provide optimal returns for the population [54]. The phenomenon of microbial biofilm formation is also related to other survival strategies like metal and antimicrobial resistance, tolerance and bioremediation [55, 56]. Application of biofilm mediated bioremediation has been found superior to other bioremediation strategies and is being applied in bioremediation of different environmental pollutants [57–61]. Microorganisms that develop a biofilm and have the ability to secrete polymers establishing a protective extracellular matrix, are physiologically more resilient to environmental changes, making them a logical choice for the treatment of different pollutants. These microbes use different strategies like biosorption, bioaccumulation and biomineralization to slowly degrade compounds [62].
Biofilm formation by Pseudomonas species had been well documented in literature[63]. P. aeruginosa is a remarkably adept opportunist with striking ability to develop biofilm [64]. In our previous study, we also observed biofilm formation by our isolate P. aeruginosa strain S3 on the surface of PLA during the process of biodegradation [12]. Phenomenon of biofilm formation on the surface of PLA had previously been reported by other authors as well [65–67].
In the genetic analysis of our isolate we found factors involved in the development of matrix of P. aeruginosa biofilm and its regulation (Table 4). The genes for three types of exopolysaccharides (EPS), previously reported as involved in construction of biofilm matrix of P. aeruginosa, (Pel, Psl and alginate [64, 68]) were found in our isolate. These EPS molecules form the protective matrix [69]. Psl is the primary factor in charge of the initiation and maintenance of biofilm structure by providing cell to cell and cell to surface interactions [70–73]. It also works as a signaling molecule to the successive events involved in the formation of biofilms and also acts as a defensive layer for different immune and antibiotic attacks [74]. Pel polysaccharide is a glucose-rich extracellular matrix and is involved in the formation of biofilms that are attached to the solid surfaces. It is considered to be less important compared to Psl [64, 73, 75, 76]. In P. aeruginosa from clinical isolates of CF patients Alginate is produced [23]. Besides its role in maintenance and protection of biofilm structure, it is essential for water and nutrient preservation [77].
Biofilm formation is a multicellular process stimulated by both environmental signals controlled by regulatory networks. During the biofilm formation, cells undergo many phenotypic shifts that are regulated by a large array of genes [78]. In the genome of P. aeruginosa strain S3, several regulatory factors were identified. One of these regulatory factors was the signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) which is considered as one of the most significant molecular determinants in biofilm regulation [79]. A c-di-GMP molecule controls the interchange between the planktonic and biofilm-associated lifestyle of bacteria by stimulating the adhesins biosynthesis and exopolysaccharides during formation of biofilm [80]. The bacterial cell to cell communication system known as quorum sensing (QS) is involved in the maintenance of many biological processes like biofilm formation, bioluminescence, antibiotics production, virulence factor expression, competence for DNA uptake, and sporulation [81, 82].
LasR/LasI, RhlR/RhlI and PQS are the three quorum sensing signaling systems employed by P. aeruginosa to control biofilm formation [83, 84]. These three QS signaling system were found to be the part of the genome for our isolate.
There is little information in the literature regarding the genetic elements involved in biofilm formation in Sphingobacterium, Genome analysis of our isolate Sphingobacterium sp., strain S2 showed presence of genes for Stage 0 sporulation protein YaaT. This protein is reported to be involved in the sporulation process and biofilm development [85, 86].
3.6. Enzymes
Biodegradation of polymers is carried out by two types of enzymes, extracellular enzymes that degrade long chain polymers into short oligomers or subunits that are subsequently carried inside the cell, and intracellular enzymes that further degrade the small transported units [87, 88]. Degradation of synthetic polymers in the environment can be a slow process [89, 90]. PLA is a synthetic linear aliphatic polyester of lactic acid monomers joined together by ester linkages [3]. The presence of ester bonds in its backbone make the polymer sensitive to hydrolysis, both chemically as well as enzymatically [88]. Biodegradation of polyesters is mostly carried out by esterolytic enzymes such as esterases, lipases, or proteases. Microbial carboxyl esterases: classification, properties and application in biocatalysis). In the literature microbial degradation of PLA is mainly reported by proteases, lipses, esterses and a few cutinases as well [4]. Both Sphingobacterium sp. and P. aeruginosa had been documented before to have a role in the degradation of different environmental pollutants such as mixed plastic waste, PAHs, oil, and dyes and pesticides, P. aeruginosa has also been reported to have potential to degrade PLA nano-composites [19, 26, 28, 91, 92]. In our previous study we characterized the degradation of PLA by our isolates [12]. Genome annotation of of both strains report presence of hydrolytic enzymes in their genomes, putatively related to their degradation of PLA (Table 5). In the genome of P. aeruginosa S3, 75 different types of proteases, 50 esterases and 25 different types of lipases were detected.
Similarly, in the genome of Sphingobacterium sp. S2 36 proteases, 30 esterases and 18 lipases were identified. Apart from these various phosphodiesterases, oxygenases, catalases and phosphatases have also been found in the genome of both isolates. Already established potential of these strains to degrade various environmental pollutants is reaffirmed by the presence of these diverse enzymes.
4. Conclusion
The present study reports the whole genome sequence analysis of two bacterial strains P. aeruginosa S3 and sphinogobacterium sp. S2, isolated from the compost and having the potential to degrade poly(lactic acid), PLA, at mesophillic temperatures (~30°C). Draft genomes of both the strains were studied to gain an insight into the genetic elements that are involved in conferring different properties to the strains helping in the degradation of PLA. The catabolic genes responsible for biodegradation of different xenobiotic compounds, genes responsible for formation and regulation of biofilm, genes for transport and utilization of lactate and several enzymes predicted to be involved in the degradation of many organic pollutants were identified on the graft genomes. All these characteristics demonstrate the degradation potential of the strains against PLA observed in the previous studies by our group; importantly it gives insight into the possible enzymes involved in the degradation of the polymer.
5. Acknowledgements
The authors show our gratitude to the Higher Education Commission of Pakistan (HEC) and the Government of Pakistan for providing a fellowship for S.S. through the international research support initiative program (IRSIP). We are also thankful to The Genomics Facility (MSU-RTSF) at Michigan State University (East Lansing, MI, USA) for performing whole genome sequencing for our strains.
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