S72 is a gram-negative rod and moves by means of a monotrichous flagellum. It is between 1.0-3.5 µm in length and 0.6-1 µm in width (Fig. S1). S72 effectively use D glucose, L arabinose, Mannose, N acetyl glucosamine, maltose, potassium gluconate, Malic acid, citrate, galactose, citrate and esculin for its growth. It however could not use mannitol, adipic acid, capric acid, phenyl acetic acid as a substrate for its growth (Table 1).
The preliminaries analysis showed that S72 can grow in the minimal media (BH) supplemented with polycyclic aromatic hydrocarbons (PAH) (biphenyl, phenanthrene, xylene, toluene, and naphthalene). The number of colonies forming units appearing on the plates decreased as the concentration (0, 20, 40, 80 100 and 150 µg/mL) of the PAH in the BH medium increased. The preliminary analysis also revealed that the S72 could better tolerate the hydrocarbons at a concentration that is ≤ 100µg/ml, but their growth in PAH began to decline at any concentration greater than 100µg/ml. S72 did not show any growth when inoculated in BH medium containing 150µg/ml toluene or xylene and showed limited growth in plate with naphthalene, phenanthrene or biphenyl at this concentration. Also, the rate at which S72 grew in medium containing toluene and xylene is lower than other PAHs tested at 100µg/ml. (Fig. 1). Based on the reported observations 100µg/ml was selected as the study concentration for the experiment.
S72 degraded all the PAHs tested at 100µg/ml with varying efficiencies. S72 showed the best activity in phenanthrene, resulting in the 93 % degradation. The degradation of biphenyl, and naphthalene lead to 85 and 81% reduction, respectively (Fig. 2). However, a low degradation efficiency was observed in the medium containing xylene (19% and 31%) respectively. The degradation was confirmed by the reduction in the peak for the PAHs as compared to the control from the GC-MS analysis (Fig 3, S2).
Genomic analysis has been used severally to unravel several details about bacterial behavior. As a result of this, the genome of S72 was sequenced and analyzed to relate its function with its genetic constituent. The sequenced genome of S. yanoikuyae S72 was reduced to 1 contig, consisting of 5,532,633 bp, 5005 CDS, and 4 identical copies of the rRNA gene operon (23S, 16S and 5S). The overall G + C content of the assembled genome is 64.23 %. The genome has 5231 putative genes and 75 RNAs (67tRNA and 12 rRNA). 1140 genes are in operons, 2515 are parts of the clusters of orthologous gene (COGs) Table2. S72 has some COG categories in abundance than some other Sphingobium species which has been previously reported. These COG categories include energy production and conversion (COG C, 6.35), carbohydrate transport and metabolism (COG G, 6.20%), lipid transport and metabolism (COG I, 6.99), secondary metabolites biosynthesis (COG Q, 4.52%), general function prediction only in transport and catabolism (COG R, 9.93) (Table 3), In a related analysis S72 was found to have higher COG abundance than 17 other hydrocarbon degrading bacteria retrieved from IMG database Table 4). It showed abundance in the COG categories Mobilome: prophages, transposons (COG X, 2.03%), Secondary metabolites biosynthesis, transport and catabolism (COG Q, 4.52%), Carbohydrate transport and metabolism (COG G, 6.20%), Lipid transport and metabolism (COG I, 6.99%), Cell wall/membrane/envelope biogenesis (COG M, 6.58), Replication, recombination and repair (COG L, 3.33%), Inorganic ion transport and metabolism (COG P, 6.22%), Transcription (COG K, 7.8), Posttranslational modification, protein turnover, chaperones ( COG, O, 4.27%), and General function prediction only (COG R, 9. 93%) Some of these COG categories have been previously reported to be associated with the degradation and mineralization of PAHs (Pal et al. 2017; Elufisan et al. 2019). The deep sequence analysis of S72 showed that it possesses 37 genes in different COG categories which are associated with the degradation of xenobiotics and polycyclic aromatic hydrocarbons. These genes include 2 pyrone-4 – 6 – decarboxylase hydrolase (A6768_17510, COG R), 4 carboxy
– 2 – hydroxy muconic semialdehyde dehydrogenase (A6768_17470, COG R). These two genes have been linked to the degradation of Benzoate and Fluorobenzoate by bacteria. Bacteria have been reported to use the enzyme encoded by these genes for the degradation of benzoate via hydroxylation (Oltmanns et al. 1989). Others include S-(hydroxymethyl) glutathione dehydrogenase/alcohol dehydrogenase (A6768_05785, COG R) that is associated with the degradation of naphthalene and chloroalkane (Yang et al. 2017). S-(hydroxymethyl) glutathione dehydrogenase/alcohol dehydrogenase has been previously reported to be associated with the degradation of naphthalene (Das et al. 2015; Pal et al. 2017). Thus, the presence of S-(hydroxymethyl) glutathione dehydrogenase/alcohol dehydrogenase in S72 could be associated with its need to degrade naphthalene as a substrate for growth. Alcohol dehydrogenase (cytochrome c) (A6768_00755, COG G), aldehyde dehydrogenase (NAD+) (A6768_10925, COG E), aldo/keto oxidoreductase (A6768_11370, COG R), Phenylacetaldehyde dehydrogenase (A6768_11850, COG C) known to participate in the degradation of fluorobenzoate toluene and naphthalene are also present in S72 (Pal et al. 2017; Elufisan et al. 2020). Other PAH degrading genes identified in S72 include propanol-preferring alcohol dehydrogenase (A6768_17370, EC:220.127.116.11), carboxymethylenebutenolidase (A6768_23915), catechol 1,2-dioxygenase (A6768_16795), muconate cycloisomerase (A6768_16805), aryl-alcohol dehydrogenase (A6768_11850), 2-keto-4-pentenoate hydratase (A6768_14055), oxalocrotonate tautomerase (A6768_04785) benzoate 1,2-dioxygenase alpha subunit (A6768_16790), benzoate/toluate 1,2-dioxygenase beta subunit (Ben B), dihydroxy cyclohexadiene carboxylate dehydrogenase (Ben D). Similarly, a gene encoding the lactoylglutathione lyase was found in S72. The lactoylglutathione lyase enzyme has been described to be actively involved in the cleavage of aromatic bond in many aromatic hydrocarbons (Mesarch et al. 2000). Among the observed genes in S72 are genes which have been reported to be specifically associated with the degradation of toluene. The genes include 4 genes encoding 3-hydroxyacyl-CoA dehydrogenase (EC:18.104.22.168) one gene for aryl-alcohol dehydrogenase (EC:22.214.171.124), and a gene for Catechol 1,2-dioxygenase (EC:126.96.36.199). Others include 2 genes encoding the enzyme oxidoreductases (EC:1.14.13.-) which often act on paired donors, with incorporation or reduction of molecular oxygen. 3 copies of the genes encoding Ferredoxin--NAD (+) reductase (EC:188.8.131.52), 7 copies of Acyltransferases (EC:2.3.1), 3 copies of carboxymethylenebutenolidase and 1 copy of muconate cycloisomerase.
S72 relatedness to other Sphingobium strain (105) was evaluated on PYANI (Pritchard et al. 2016) using the average nucleotide identity mummer (ANIm) comparison measure. The ANIm result showed that S72 is closely related to S. yanoikuyae strain UBA2097 sharing 97% average identity with it (Fig. 4). The analysis of S72 pan genome showed that it shared 1734 core genome with other Sphingobium species and possess 403 unique genes. We noted that 126 of the unique genes are associated with the catabolism of xenobiotics. Twenty out of the unique genes have been previously reported to be involved in the degradation of toluene, xylene, ethylbenzene, biphenyl, benzoate, naphthalene, anthracene, tetrachloroethene, 1, 4-dichlorobenzene, bisphenol, trinitrotoluene in bacteria. These genes are involved in both the catabolism of central aromatic intermediate and the peripheral catabolic pathway for aromatic hydrocarbon. (Table S1).
Horizontal gene transfer is a common phenomenon through which bacteria often acquire some genes that are essential for their survival (Elufisan et al. 2020). The acquired genes are commonly found on the genomic islands in bacteria or as mobile genetic elements. The analysis of the S72’s genome with the online based Island viewer 4.0, revealed that it has 37 genomic islands (Fig. 5). Five large regions were found in the genomic Island with regions II and III being the largest consisting of 317,308 kb and 579,060 kb in length, respectively. An in-depth look into the genomic island showed the presence of many genes that are associated with the degradation of xenobiotics and PAHs. Among such genes are SDR family NAD(P)-dependent oxidoreductase (A6768_07840), aldo/keto reductase (A6768_07825), cytochrome P450 (A6768_11830), 4-hydroxybenzoate 3-monooxygenase (A6768_12975), and aromatic alcohol reductase (A6768_RS13080). The other PAH degrading genes found on the genomic island can be seen in supplementary file.