Complete genome sequence of esterase-producing bacterium Croceicoccus marinus E4A9T

Croceicoccus marinus E4A9Twas isolated from deep-sea sediment collected from the East Pacific polymetallic nodule area. The strain is able to produce esterase, which is widely used in the food, perfume, cosmetic, chemical, agricultural and pharmaceutical industries. Here we describe the characteristics of strain E4A9, including the genome sequence and annotation, presence of esterases, and metabolic pathways of the organism. The genome of strain E4A9T comprises 4,109,188 bp, with one chromosome (3,001,363 bp) and two large circular plasmids (761,621 bp and 346,204 bp, respectively). Complete genome contains 3653 coding sequences, 48 tRNAs, two operons of 16S–23S-5S rRNA gene and three ncRNAs. Strain E4A9T encodes 10 genes related to esterase, and three of the esterases (E3, E6 and E10) was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form, revealing its potential application in biotechnological industry. Moreover, the genome provides clues of metabolic pathways of strain E4A9T, reflecting its adaptations to the ambient environment. The genome sequence of C. marinus E4A9T now provides the fundamental information for future studies.

Croceicoccus [6], as a genus of the family Erythrobacteraceae [7], can be found in the marine environments, including deep-sea sediment, surface seawater and marine biofilm from a boat shell [6,8,9]. C. marinus E4A9 T , the type strain of the genus Croceicoccus, was isolated from deep-sea sediment collected from the East Pacific polymetallic nodule area [6]. The strain was able to produce esterase as well as lipase [6]. To get insight into the capability of esterase production, recently, we obtained the complete genome of C. marinus E4A9 T and detected genes of esterase. This is the first genome report for the strain in the genus of Croceicoccus. We also describe the genomic sequencing related to its annotation for understanding their metabolic and ecological functions in the environment.

Organism information
Classification and features C. marinus E4A9 T was isolated from a deep-sea sediment sample collected from the East Pacific polymetallic nodule area (8°22′38" N, 145°23′56" W) at a depth of 5280 m (temperature 2°C, salinity 3.4%). Strain E4A9 T was obtained and routinely cultured on marine broth 2216 (MB, BD) at 30°C. Subsequently polyphasic study of strain E4A9 T was performed. A new species Croceicoccus marinus gen. Nov. sp. nov. was proposed. Strain E4A9 T is the type strain of the species of C. marinus [6], and was deposited into the China General Microbiological Culture Collection (CGMCC 1.6776 T ).
C. marinus [6] is a valid species belonging to the family Erythrobacteraceae [7], in the order Sphingomonadales [10,11], class Alphaproteobacteria [11,12] and phylum Proteobacteria [13] .C. marinus E4A9 T is a Gramstaining-negative and cocci-shaped bacterium (Fig. 1). It grew aerobically and used a series of organic carbon, such as L -arabinose, D -cellobiose, D -galactose and xylose, as sole sources of carbon and energy [6,8]. Based on phylogenetic analysis of 16S rRNA gene sequence, the strain falls into the cluster comprising the Croceicoccus species with a high bootstrap value (Fig. 2). Interestingly, strain E4A9 T could hydrolyze Tween 20, Tween 80 and tributyrin, indicating the presence of esterase as well as lipase [6]. The API ZYM system also supported the results that esterase (C4) and esterase lipase (C8) activities are present.
The general features of strain E4A9 T was summarized in Table 1.

Genome sequencing information
Genome project history C. marinus E4A9 T [6] was selected for sequencing because it is relevant to genomic sequencing of the whole family of Erythrobacteraceae [7] and esterase production. The complete genome sequence was finished on May 29, 2015. The gap closure and annotation processes were performed by the authors. The GenBank accession number of the genome is CP019602, CP019603 and CP019604. The main genome sequence information is present in Table 2 and Table 3.
Growth conditions and genomic DNA preparation C. marinus E4A9 T was aerobically cultivated in Marine Broth (MB, BD Difco™) at 30°C and stored at −80°C   a Evidence codes -IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [32]  with 30% (v/v) glycerol. High-quality genomic DNA was extracted using the Qiagen DNA extraction kit, according to its protocol.

Genome sequencing and assembly
The genome of strain E4A9 T was sequenced using SMRT technology with a PacBio RS II platform (Zhejiang Tianke Co. Ltd., China). One library was constructed with 10 kb insert size according to the large SMRTbell gDNA protocol (Pacific Biosciences, USA). The sequencing generated 85,372 reads with an average length of 11,938 nt (972 Mb, 248-fold genome coverage). The de novo assembly of the reads was performed using HGAP Assembly version 2 (Pacific Biosciences, USA). The circularization of final contigs was checked and the overlapping ends were trimmed.

Genome annotation
The rRNA genes were found via RNAmmer 1.2 Server [14] and tRNA genes were identified using tRNAscan-SE 2.0 online server [15]. The open reading frames (ORFs) and the functional annotation of translated ORFs were performed using the RAST server online [16] and GeneMarkS+. Classification of some predicted genes were analyzed using COG database [17] and Pfam [18]. Genes with signal peptides were predicted using SignalIP 4.1 Server [19]. Genes with transmembrane helices were performed using TMHMM Server v. 2.0 [20]. The clustered regularly interspaced short palindromic repeats structures of the genomes were searched by CRISPRfiner program online [21]. Translated genes were assigned to Kyoto Encyclopedia of Genes and Genomes pathway using KEGG automatic annotation server with BBH method [22,23]. The circular map of chromosome and plasmids were obtained using a CG View online server [24].

Genome properties
The general features of strain E4A9 information are displayed in Table 1 and  (Fig. 3). The G + C content was 64.5 mol%. The genome of strain E4A9 contains 3653 coding sequences (CDSs), 48 tRNAs, two operons of 16S-23S-5S rRNA gene and three ncRNAs. Among the genes, 132 were assigned to pseudogene. The summary of features and statistics of the genome is shown in Table 4 and genes belonging to COG functional categories are listed in Table 5. Three replicons of the genome of strain E4A9, located in a circular chromosome and two large plasmids, were detected. Two plasmid replication initiator protein genes (ARU17925 and ARU18299) were found in the two plasmid sequence respectively, indicating that the genome of strain E4A9 contains two large circular plasmids. The G + C content of the two plasmids (63.5 mol% and 60.7 mol%, respectively) was a litter lower than that of the chromosome (65.2 mol%). The two plasmids have high gene density with 702 and 303 protein-coding regions, respectively. Many unsuspected genes involved in metabolism of aromatic compounds were identified in plasmid pCME4A9I. Almost 10% of the plasmid pCME4A9II sequence carries genes encoding gene of subsystem feature virulence, disease and defense, and most of them were of the copper homeostasis and cobalt-zinc-cadmium resistance. The functions of these genes are consistent with the notion that the two plasmids play an important role in the adaption of the bacteria in the sediment environment.

Insights from the genome sequence
Esterases presence of C. marinus E4A9 T The presence of genes for the biotechnologically important enzymes like lipolytic enzymes were also predicted. Ten novel esterases were predicated (Fig. 4), and their amino acid sequences shared 58% to 85% identities to those of other lipolytic enzymes in the database. Phylogenetic analysis showed that predicated esterases E3 and E6 were grouped into family VII lipolytic enzymes and E10 was grouped into family II lipolytic enzymes. In order to investigate the biochemical properties of the esterases (E3, E6 and E10), recombinant plasmids were constructed and expressed in Escherichia Coli [25,26]. After incubation of recombinant colonies for 48 h on the plate (Luria-Bertani agar medium) supplemented with 1% tributyrin, the three recombinant colonies had clear zones around the colonies. It indicated the presence of lipolytic activity. The calculated molecular weight of E3, E6 and E10 was 55.9, 46.1 and 22.4 kDa, respectively. The recombinant protein was soluble and purified using a Ni-NTA affinity chromatography column. The activity of purified E3, E6 and E10 was examined using p-nitrophenyl butyrate as substrate, and they had specific activities under standard reaction conditions (data not shown).

Metabolism of C. marinus E4A9 T
The complete genome of C. marinus E4A9 T was annotated for understanding the metabolic potentials based on the key genes of metabolic pathways of carbon, nitrogen, sulfur and phosphorus. (i) Carbon metabolism. The genome of strain E4A9 T is lack of carbon fixation and CO-oxidizing (cox) genes, indicating that the strain is not able to grow autotrophically. Strain E4A9 T can use organic carbon sources ( Table 1). The genome has a complete glycolysis pathway (Embden-Meyerhoff-Parnas . From outside to the center: CDSs and RNA genes on the forward strand (colored by COG categories), CDSs and RNA genes on the reverse strand (colored by COG categories), G + C content (peaks out/inside the circle indicate values higher or lower than the average G + C content, respectively), GC skew (calculated as (G-C)/(G + C), green/purple peaks out/inside the circle indicate values higher or lower than 1, respectively), genome size pathway). In addition, it possesses key genes of the Entener-Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. (ii) Nitrogen metabolism. The genome of C. marinus E4A9 T possesses ammonium transporter genes and amino acids transporter genes (e.g. methionine and L-proline/glycine betaine). Genes encoding enzymes involved in polyamines biosynthesis are present, but the lack of polyamines transporters suggests its incapability of utilizing extracellular polyamines. Nitrate and nitrite transporters have been found in the genome of strain E4A9. It processes genes involved in nitrate and nitrite reduction (nasAB and nirBD, respectively) and is lack of genes involved in denitrification, nitrogen fixation and anammox. Thus, nitrate and nitrite could act as electron acceptors to generate ammonium, subsequently being utilized by strain E4A9 as a reduced nitrogen source.
The genome of C. marinus E4A9 T is lack of urease (ureABC); however it harbors genes involved in urea decomposition, including urea carboxylase-related ABC transporter, urea carboxylase-related aminomethyltransferase, urea carboxylase and allophanate hydrolase, suggesting its capability of utilizing urea as a C or N source in the  The total is based on the total number of protein coding genes in the genome environment [27]. (iii) Sulfur metabolism. Strain E4A9 T possesses genes involved in assimilatory sulfate reduction (e.g. cysND, cysC, cysH, cysJI). Sulfate can be reduced to sulfide, subsequently being incorporated into amino acids. Genes involving in alkanesulfonate assimilation (arylsulfatase and FMN reductase) are present in the genome of strain E4A9, suggesting its capability of utilizing organic sulfur compounds. However, it missed transporter genes for the uptake of extracellular alkanesulfonates. (iv) Phosphorus metabolism. Strain E4A9 is lack of genes for inorganic P storage as polyphosphate (ppk), as well as transport (phnCDE) and cleavage (phnGHIJKLN) of organic P in the form of phosphonates [28]. While strain E4A9 possesses the high-affinity phosphate transport system (pstSCAB) and regulatory genes (phoUBR), indicating an alternative strategy for maintaining a reliable supply of phosphorus [29].

Conclusions
The complete genome sequence of C. marinus E4A9 T contains a circular chromosome as well as two large circular plasmids and provides an insight into the genomic basis of its esterases production ability. Our data implies C. marinus E4A9 T is a potential candidate in biotechnological application and facilitates the understanding for further industrial and biotechnological applications of esterases.
Abbreviations CDS: Coding sequence; CRISPRs: Clustered regularly interspaced short palindromic repeats; KAAS: KEGG automatic annotation server; KEGG: Kyoto encyclopedia of genes and genomes; ORF: Open reading frame