Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity

Abstract

Background: Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate.

Results: In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity.

Conclusions: Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates.

Fig. 1

a A representative plate from primary high-throughput screening with three potentially laccase-positive hits indicated with arrows. b A representative plate from the laccase-activity confirmation step

Fig. 2

Phylogenetic tree of laccase-positive Psychrobacter species based on 16S rRNA gene sequence similarity. Psychrobacter strains with laccase-positive phenotype are indicated in bold font. Psychrobacter type strains were used as references, with Moraxella atlantae as an outgroup. The four strains selected for genome sequence determination are indicated with arrows

Fig. 3

Hierarchical genome clustering of Psychrobacter species based on KEGG pathways. The four genome sequenced Psychrobacter species in this study are indicated in bold font and with arrows

Fig. 4

Genome clustering of Psychrobacter species based on KEGG pathways by principal component analysis (PCA). The four genome sequenced Psychrobacter species in this study are indicated with red spheres and arrows

Fig. 5

Gene organizations of gene candidates and their positions. 1: LMCO; 2: copB; 3: ATP-binding protein; 4: Cytochrome C; 5: amino acid permease; 6: Isoprenylcysteine carboxyl methyltransferase; 7: Copper-transporting P-type ATPase; 8: Oxidoreductase; Grey arrows represents hypothetical proteins

Fig. 6

Enzymatic assay of P11F6-LMCO and P11G5-LMCO2 using ABTS and guaiacol as substrates. Increase in A ₄₂₀ and A ₄₆₈ when ABTS (a) and guaiacol (b) were used as the substrates, respectively. c Chromogenic products of ABTS and guaiacol oxidation by the studied LMCOs