Salt Adaptation and Evolutionary Implication of a Nah-related PAHs Dioxygenase cloned from a Halophilic Phenanthrene Degrading Consortium

Abstract

Polycyclic aromatic hydrocarbons (PAHs) pollutions often occur in marine and other saline environment, largely due to anthropogenic activities. However, study of the PAHs-degradation genotypes in halophiles is limited, compared with the mesophilic terrestrial PAHs degraders. In this study, a bacterial consortium (CY-1) was enriched from saline soil contaminated with crude oil using phenanthrene as the sole carbon source at 10% salinity. CY-1 was dominated by the moderate halophilic Marinobacter species, and its dominant PAHs ring-hydroxylating dioxygenase (RHD) genotypes shared high identity to the classic nah-related RHDs found in the mesophilic species. Further cloning of a 5.6-kb gene cluster from CY-1 unveiled the existence of a new type of PAHs degradation gene cluster (hpah), which most probably evolves from the nah-related gene clusters. Expression of the RHD in this gene cluster in E. coli lead to the discovery of its prominent salt-tolerant properties compared with two RHDs from mesophiles. As a common structural feature shared by all halophilic and halotolerant enzymes, higher abundance of acidic amino acids was also found on the surface of this RHD than its closest nah-related alleles. These results suggest evolution towards saline adaptation occurred after horizontal transfer of this hpah gene cluster into the halophiles.

Figure 1

Phylogenetic Neighbor-Joining tree showing the V1 to V3 region of the 16 S rRNA gene sequences of CY-1. The sequences data is separated into different OTUs with 97% identity. The proportions of different genera in CY-1 are shown following the OUT names, and the three most abundant OTUs are shown in bold. For each genus present in the consortium, a reference sequence is included in the phylogenetic tree, and the accession number is shown after the specie names. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. 16 s rRNA gene sequence from Methanococcus Vannielii is used as the outgroup in the tree.

Figure 2

Maximum Likelihood tree of the representative pahAc of the dominant genotype in CY-1, the full length pahAc gene in the newly-cloned hpah gene cluster and other representative nah-related Ac genes, built with predicted amino acid sequences. Several characterized pahAcs from Alteromonas sp. SN2, Burkerholderia sp. RP007, Sphingomonas sp. B1 and Rhodococcus sp. NCIB12308 were used as outgroup. PahAc sequences cloned in this study are shown in bold. The five types of the nah-related pahAcs are labeled in the tree, with the group’s name shown in the right of the tree.

Figure 3

Concatenated tree of nah-related AbAcAdB genes, organization of nah-related gene clusters and proposed evolutionary scenario. On the left is the concatenated tree of nah-related AbAcAdB genes deduced by both the Maximum Likelihood and Neighbor-Joining method; AcAdB genes from Alteromonas sp. SN2 were used as the outgroup. On the right are the archetypal structures of the five types of the nah-related gene clusters. Arrows in white represent PAHs degradation genes and their transcription directions. For clarity, nahAaAbAcAd are shown as a, b, Ac, d, etc. Arrows in gray represent genes encoding hypothetical proteins. Arrows with dashed borders represent interrupted genes. The proposed evolutionary events are shown next to the corresponding branches in the tree.

Figure 4

Effects of NaCl concentration on the activity (A) and salinity stability (D) of RHD obtained from CY-1 and the effect of NaCl concentration on the RHD activity from a non-halotolerant species, Pseudomonas (B) and Delftia sp. Cs1–4 (C). Effects of NaCl concentration (A, B and C) was determined at 4 °C in 50 mM PBS buffer (pH = 7.5). Activity was detected by the standard method. The value obtained without NaCl in the reaction mixture was taken as 100%.The enzyme was incubated in sodium phosphate buffers (pH 7.5) and PBS contain 5%, 10%, 15%, 20% NaCl respectively to detect the RHD salinity stability (D) and residual activity was determined at 3, 6, 9, 12, 24 h. The values shown represent averages from triplicate experiments. Error bars were not shown in this fig for better looking effect.

Figure 5

Surface electrostatic potentials analysis. (A) Surface electrostatic potentials of nahA3, and Naphthalene 1,2-dioxygenase from Pseudomonas putida NCIB 9816–4 (PDB no. 1O7N) as obtained using Discovery Studio 2.5 software. The first, second and third line were seen from the front, top and left of the structures, respectively, with the red surface corresponding to negatively charged residues and the blue surface corresponding to positively charged residues (color figure online). (B) The key amino acid sites in nahAc. Fe²⁺ binding sites are labelled in black squares and the sequence near these active sites are also shown. Mutated amino acid sites are labelled in red frame. (C) The position of these mutated sites appear at the surface of the large subunit. This graph is illustrated by Swiss-pdb viewer.

Figure 6

Neighbour-Joining trees for nagA1 (1), nagA2 (2), nagG (3), nagH (4) and their homologous genes. Branches in red are from the nah-related gene clusters; branches in bold black are from the Sal5H gene clusters; branches in green are from some representative Sal5H gene clusters whose structures are present in Fig. S7.