Fig 1.
Characterization of Alcaligenes aquatilis BU33N.
A) Colonies of BU33N on TSA medium B) Biosurfactant production (hemolytic activity); C) Biosurfactant production [Blue agar (CTAB) method]; D) emulsification activity (E24 test), E) Scanning electron micrographs of strain BU33N grown on TSA medium (at 28±1°C for 48h).
Table 1.
Classification and general features of Alcaligenes aquatilis BU33N.
Fig 2.
Phylogenetic analysis of 16S rRNA gene sequence of bacterial isolate Alcaligenes aquatilis strain BU33N.
The maximum likelihood (ML) tree was generated based on the alignment of the 16s RNA genes of BU33N, QD168 and the type species of the genus Alcaligenes. The ML was constructed based on the TN+F+G4 model using IQ-TREE with confidence values based on 1000 bootstrap replicates. Burkholderia cepacia was used to position the root of the tree.
Fig 3.
Degradation rate of hydrocarbon by BU33N.
A) Relative percentage (%) of abundance (presence) of n-alkanes (C12—C36) after 21 days of incubation by BU33N; B) Degradation rate (%, residual hydrocarbons) of crude oil (TERCHs and n-alkanes) and phenanthrene by Alcaligenes aquatilis BU33N after 21 days of incubation.
Fig 4.
BLAST comparison of draft genome of Alcaligenes aquatilis against three Alcaligenes species, using GC View.
The innermost rings depict GC content (Black) and GC Skew (purple/green) followed by concentric rings of query sequences colored according to BLAST identity. The outermost rings depict genomes of the following microbes Alcaligenes aquatilis QD168 (Pink), Alcaligenes faecalis (Green) and Alcaligenes faecalis subsp. phenolicus (Blue).
Fig 5.
The benzoate (A) and catechol (B) biodegradation pathway in BU33N: A (i) benzoate 1, 2-dioxygenase alpha subunit (EC 1.14.12.10), (ii) beta subunit (EC 1.14.12.10), (iii) Ortho-halobenzoate 1, 2-dioxygenase alpha-ISP protein OhbA, (iv) beta-ISP protein OhbB (v) by 1, 2-dihydroxycyclohexa-3, 5-diene-1-carboxylate dehydrogenase (EC 1.3.1.25).
B (vi) catechol 1, 2-dioxygenase (EC 1.13.11.1), (vii) muconatecycloisomerase (EC 5.5.1.1), (viii) muconolactone isomerase (EC 5.3.3.4), (ix) beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24), (x) 3-oxoadipate CoA-transferase subunit A (EC 2.8.3.6), (xi) 3-oxoadipate CoA-transferase subunit B (EC 2.8.3.6) and (xii) beta-ketoadipyl CoA thiolase (EC 2.3.1.-).
Fig 6.
Gene organization of the first benzoate degradation enzymes encoding gene cluster in Alcaligenes aquatilis BU33N compared to that of selected hydrocarbon degrading bacteria.
The strain identities, BU33N, QD168, BDB4, DR1, ATCC 49840 and LMG 23572 represent the bacteria Alcaligenes aquatilis BU33N, Alcaligenes aquatilis QD168, Alcaligenes faecalis BDB4, Acinetobacter oleivorans DR1, Marinobacter hydrocarbonoclasticus ATCC 49840 and Pseudomonas xanthomarina LMG 23572, respectively.
Fig 7.
Gene organization of the first catechol degradation enzymes encoding gene cluster in Alcaligenes aquatilis BU33N compared to that of selected hydrocarbon degrading bacteria.
The strain identities, BU33N, QD168, BDB4, DR1, ATCC 49840 and LMG 23572 represent the bacteria Alcaligenes aquatilis BU33N, Alcaligenes. aquatilis QD168, Alcaligenes faecalis BDB4, Acinetobacter oleivorans DR1, Marinobacter hydrocarbonoclasticus ATCC 49840 and Pseudomonas xanthomarina LMG 23572, respectively.
Fig 8.
The A) phenanthrene degradation and B) β-ketoadipate pathways in Alcaligenes aquatilis BU33N.
(I) Phenanthrene, (II) cis-3,4-Dihydroxy-3,4-dihydrophenanthrene, (III) 3,4-Dihydroxyphenanthrene, (IV) 2-Hydroxy-2H-benzo[h]chromene-2-carboxylate, (V) trans-4-(1'-Hydroxynaphth-2'-yl)-2-oxobut-3-enoate, (VI) 1-Hydroxy-2-naphthaldehyde, (VII) 1-Hydroxy-2-naphthoate, (VIII) trans-2'-Carboxybenzalpyruvate, (IX) 2-Carboxybenzaldehyde, (X) Phthalate, (XI) Phthalate 3,4-cis-dihydrodiol, (XII) 3,4-Dihydroxyphthalate, (XIII) 3,4-Dihydroxybenzoate, (XIV) 3-Carboxy-cis,cis-muconate, (XV) 4-carboxymuconolactone, (XVI) ß-ketoadipate enol-lactone and (XVII) 3-Oxoadipic acid; Beta-ketoadipate. Predicted enzymes include, (Ea) Ring hydroxylating dioxygenase, alpha subunit (EC 1.14.12.13), (Eb) 3-phenylpropionate dioxygenase beta subunit (EC 1.14.1.-), (Ec) 1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (EC 1.3.1.25), (Ed) Biphenyl-2,3-diol 1,2-dioxygenase (EC 1.13.11.39), (Ef) Maleylacetoacetate isomerase (EC 5.2.1.2), (Eg) Possible carboxymuconolactone decarboxylase family protein (EC 4.1.1.44), (Eh) 4-hydroxy-2-oxovalerate aldolase (EC 4.1.3.39), (Ei) 4-carboxymuconolactone decarboxylase (EC 4.1.1.44), (Ej) Acetaldehyde dehydrogenase (EC 1.2.1.10), (Ek) Acetaldehyde dehydrogenase, acetylating, (EC 1.2.1.10), (El) carboxymuconolactone decarboxylase, (Em) Aldehyde dehydrogenase (EC 1.2.1.3), (En) Benzoate 1,2-dioxygenase beta subunit (EC 1.14.12.10), (Eo) Benzoate 1,2-dioxygenase alpha subunit (EC 1.14.12.10), (Ep) Muconatecycloisomerase (EC 5.5.1.1), (Eq) Homogentisate 1,2-dioxygenase (EC 1.13.11.5), (Er) Ortho-halobenzoate 1,2-dioxygenase beta-ISP protein OhbA, (Es) Ortho-halobenzoate 1,2-dioxygenase alpha-ISP protein OhbB, (Ep) Muconatecycloisomerase (EC 5.5.1.1), (Et) carboxymuconolactone decarboxylase and (Eu) Beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24).