Genome Analysis of the Marine Bacterium Labrenzia sp. Strain 011, a Potential Protective Agent of Mollusks

Abstract

The marine bacterium Labrenzia sp. strain 011 was isolated from the coastal sediment of Kronsgaard, Germany. The Labrenzia species are suggested to be protective agents of mollusks. Labrenzia sp. strain 011 produces specialized metabolites, which showed activity against a range of microorganisms, thereunder strong inhibitory effects against Pseudoroseovarius crassostreae DSM 16,950 (genus Roseovarius), the causative agent of oyster disease. The genome of Labrenzia sp. strain 011 was sequenced and assembled into 65 contigs, has a size of 5.1 Mbp, and a G+C content of 61.6%. A comparative genome analysis defined Labrenzia sp. strain 011 as a distinct new species within the genus Labrenzia, whereby 44% of the genome was contributed to the Labrenzia core genome. The genomic data provided here is expected to contribute to a deeper understanding of the mollusk-protective role of Labrenzia spp.

Dataset: This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. QCYM00000000. The version described in this paper is the first version, QCYM01000000 (https://www.ncbi.nlm.nih.gov/nuccore/QCYM01000000).

Dataset License: CC0 (databases of molecular data on the NCBI Web site include examples such as nucleotide sequences (GenBank), protein sequences, macromolecular structures, molecular variation, gene expression, and mapping data. They are designed to provide and encourage access within the scientific community to sources of current and comprehensive information. Therefore, NCBI itself places no restrictions on the use or distribution of the data contained therein).

1. Summary

Bacteria of the genus Labrenzia colonize surfaces, such as oyster shells, and may produce antibacterial compounds, which inhibit the growth of other bacteria [1,2,3,4]. Labrenzia sp. strain 011 showed activity against the oyster pathogen Roseovarius crassostreae [1]. R. crassostreae has an adverse effect on natural oyster populations and on oyster farming operations [5]. In addition, strains of the genus Labrenzia, which produce compounds showing antimicrobial activity, were associated with soft corals and the marine sponge Erylus discophorus [6,7]. Moreover, an analysis of the available genome of Labrenzia sp. strain EL143 showed many genes that are linked to the symbiotic relationship with sessile hosts, genes that can be linked to resistance mechanisms against antibiotics and toxic compounds, and genes corresponding to a strong dehalogenation potential [8]. This can be regarded as a requirement for filter-feeding organisms that are exposed to halogenated substances in their environment, and might use bacterial symbionts with dehalogenase activity for detoxification and nutrition [9]. These reports reflect the importance of Labrenzia species and their potential for the protection of marine bivalves and for biotechnological applications. Therefore, the genome of Labrenzia sp. strain 011 will enable the identification of biosynthetic gene clusters corresponding to protective compounds. The data shown here can be useful for research groups working on natural product discovery, by enabling further genome-mining approaches.

2. Data Description

The draft genome sequence of Labrenzia sp. strain 011 consists of 65 contigs (>1000 bp) with 5,102,962 bp in length, and a G+C content of 61.6%. There were 4812 coding sequences (CDSs) that were predicted (this number includes proteins annotated as hypothetical), of which 2280 CDSs (48%) were categorized in 473 different subsystems with identified functional roles.

A phylogenetic tree of all of the Labrenzia strains with the available genomes based on the core genomes alignment revealed Labrenzia sp. strain OB1 and L. marina DSM 17,023, which were isolated from coastal seawater in La Jolla, CA, USA, and South Korea, respectively, as the most closely related strains to Labrenzia sp. strain 011 (Figure 1).

In order to obtain further insight into the degree of similarity between the analyzed genomes, the numbers of the core genes and of the singletons were determined. There were 2131 CDS that contributed to the core genome of the Labrenzia strains, equivalent to ~44% of the Labrenzia sp. strain 011 genome (Figure 2A). To identify the actual core genome of a species, it is possible to use an approximate approach by extrapolating the number of core genes for an infinite number of genomes [10]. Using this methodology, it was calculated that the core genome will be around 2113 CDS, based on a decay function (2929.005 × exp(−x/3.229) + 2112.783, see Figure 2B). The pan genome increases with every additional Labrenzia strain, indicating an open pan genome of Labrenzia (Heaps’ law function: 5736.13 × x^0.462, see Figure 2C).

The average nucleotide identity (ANI) values between Labrenzia sp. strain 011 and all of the analyzed Labrenzia strains was between 73.55% to 84.85% in the pair-wise sequence comparisons (Figure 3). This puts the strain only into distant relation to other strains, as values smaller than 80–85% ANI must be regarded as distantly related [11]. The in-silico DNA–DNA hybridization (isDDH) values between Labrenzia sp. strain 011 and the other Labrenzia strains was between 22.7% to 33.1%, whereby the highest values were obtained for Labrenzia sp. strain OB1 and L. marina DSM 17023, verifying the phylogenetic relationship between these two and strain 011. Furthermore, differences in the G+C content between Labrenzia sp. strain 011 and other Labrenzia strains were between 1.32–5.38%, which supports the species delineation (

Table 1. In silico DNA–DNA hybridization (isDDH) and G+C difference of Labrenzia sp. strain 011 vs. other Labrenzia strains.

Labrenzia sp. Strain 011 vs.	isDDH%	G+C Difference%
Labrenzia sp. strain OB1	33.1	2.20
Labrenzia marina	30.2	1.41
Labrenzia sp. strain C1B70	26.7	2.71
Labrenzia sp. strain C1B10	26.7	2.71
Labrenzia sp. strain CP4	26.6	2.52
Labrenzia sp. strain VG12	26.5	1.62
Labrenzia aggregata	26.5	2.57
Labrenzia sp. strainUBA4493	26.3	2.56
Labrenzia sp. strain DG1229	24.8	5.38
Labrenzia alba	24.4	5.25
Labrenzia alexandrii	23.5	5.26
Labrenzia suaedae	22.7	1.32

). Therefore, the in silico parameters (ANI ≥ 96%, isDDH ≥ 70%, and difference in G+C content of ≤ 1%) [11,12,13] define Labrenzia sp. strain 011 as a distinct new species of the genus Labrenzia (Figure 3, Table 1). Instead, CP4, UBA4493, C1B70, and C1B10 seem to be strains closely related to L. aggregata RMAR6, with ANI values between 97–100% (Figure 3).

The genome of Labrenzia sp. strain 011 carries genes related to nitrogen metabolism and denitrification (56 CDSs), polyhydroxybutyrate metabolism (32 CDSs), and many genes that are related to stress response, for example, heat and cold shock (169 CDSs) (Figure 4). Labrenzia sp. strain 011 belongs to the family of Rhodobacteraceae, which is a sister family of the Rhizobiales. The latter fix nitrogen in plant roots [14]. This data may explain the denitrification ability of the oyster microbiome, which is dominated by Rhodobacteraceae [15]. The bacteria of this family are surface colonizers and are known for the production of compounds with antibacterial activity, which prohibit the growth of other bacteria; thereby, shaping the microbiome [15,16].

In total, 11 biosynthetic gene clusters (BGCs) were identified (5.3% of the genome), including one type-I polyketide synthase, one terpene, one bacteriocin, four fatty acids, and four saccharide BGCs (Figure 5). Additionally, 23 putative gene clusters were identified using the cluster finder algorithm (3.8% of the genome), thereunder three BGCs for cyclopropane fatty acid synthases (Figure 5).

3. Methods

3.1. Sequencing and Assembly

The marine bacterium Labrenzia sp. strain 011 was isolated from sediment from the coastal area of Kronsgaard, Germany. The phenotypic appearance of its colonies is creamy yellow on Difco^TM marine agar 2216 (

Table 2. Features of Labrenzia sp. strain 011, and MIGS mandatory information.

Items	Description
Investigation type	Bacteria
Strain	Labrenzia sp. 011
Gram stain	Negative
Cell shape	Rod
Pigmentation	Creamy yellow
Temperature optimum	30 °C
Latitude and longitude	54.731111 N 9.964167 E
Geographic location name	Kronsgaard, Germany
Collection date	15-Aug-2012
Environmental biome	M arine biome (ENVO:00000447)
Environmental feature	Sea coast (ENVO:00000303)
Environmental material	Marine sediment (ENVO_03000033)
Environmental package	Surface sediment
Relationship to oxygen	Aerobe
Number of replicons	1
Sequencing method	Illumina

). The genomic DNA isolation of Labrenzia sp. strain 011 was performed as described before [17]. In brief, a one-week culture in a marine broth liquid medium was used to harvest the cell pellets. Therefrom, the DNA was isolated using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich). Illumina shotgun paired-end sequencing libraries were generated and sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA). Quality filtering using Trimmomatic version 0.36(6) resulted in 495,158 paired-end reads for Labrenzia sp. strain 011. The paired-end reads were combined using the Spades assembler v3.10, yielding initial sequence scaffolds [18]. Scaffolds smaller than 1 kb were filtered and 65 contigs remained as determined with Quast [19]. The genome completeness was estimated using CheckM [20] and the genus level marker genes, resulting in a value of 83.2%.

3.2. Genome Annotation and Comparison

The coding sequences (CDS) of the genome were determined using the RAST prokaryotic genome annotation server [21]. The annotated GenBank file was uploaded to the EDGAR 2.2 genomic pipeline [22] for phylogeny and genome comparison. For this analysis, all of the available genome sequences of the Labrenzia strains were used (accession numbers in parentheses), as follows: L. alexandrii DFL-11^T (ACCU00000000), L. aggregata RMAR6-6 chromosome (CP019630), L. suaedae DSM 22153^T (FRBW00000000), L. alba CECT 5095^T (CXWE00000000), Labrenzia sp. strain CP4 (CP011927), Labrenzia sp. strain VG12 (CP022529), Labrenzia sp. strain DG1229 (AYYG00000000), Labrenzia sp. strain C1B10 (AXBY00000000), Labrenzia sp. strain C1B70 (AXCE00000000), Labrenzia sp. strain OB1 (JSEP00000000), L. marina DSM 17023^T (PPCN00000000), and Labrenzia sp. strain UBA4493 (DGNL00000000). For the in silico comparison of the strains, the average nucleotide identity (ANI) matrix of all of the conserved genes in the core genome was computed by the BLAST algorithm using JSpeciesWS [23], and was visualized as heat map. The in-silico DNA–DNA hybridization (isDDH) was performed based on the identities/high-scoring segment pairs (HSP) length formula using the DSMZ genome to the genome distance calculator (GGDC) service tool [12]. The biosynthetic gene clusters (BGCs) for the specialized metabolites were identified using antiSMASH v4 [24], and default parameters and the incorporation of the ClusterFinder algorithm were applied.