Whole-genome sequence data and analysis of Lactobacillus delbrueckii subsp. lactis ACA-DC 178 isolated from Greek Kasseri cheese.

Lactobacillus delbrueckii subsp. lactis is employed in the production of various types of cheese. Here, we report the complete genome sequence of L. lactis ACA-DC 178 isolated from Greek Kasseri cheese. The chromosome of ACA-DC 178 contains 2,050,316 bp with a GC content of 49.6%. A total of 2,112 genes were identified in the genome sequence including 1,752 protein-coding genes, 239 putative pseudogenes, 94 tRNA and 27 rRNA genes. According to the COG annotation, about 80% of the protein-coding genes (1,417 proteins) were assigned to at least one functional category. Approximately the 1/3 of these proteins were distributed among three categories, namely replication, recombination and repair (category L: 10.6%), translation, ribosomal structure and biogenesis (category J: 7.5%) and amino acid transport and metabolism (category E: 7.2%). Fourteen integrated GIs with a total of 159 genes were found in ACA-DC 178 genome. Several of these genes encode proteins associated with exopolysaccharide biosynthesis, amino acid transport and subunits of restriction-modification systems. One large CRISPR array of 3,197 bp containing 52 spacers, several of which are identical to phage sequences having hosts in the genus Lactobacillus, was also identified. The annotated genome sequence of L. lactis ACA-DC 178 is deposited at the European Nucleotide Archive under the accession number LS991409. Raw reads are deposited in the Sequence Read Archive (SRR8866601-3).

Data

In this study, we present the complete genome sequence of L. lactis ACA-DC 178 isolated from Greek Kasseri cheese [1], [2]. The L. delbrueckii species consists of six subspecies, including Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunkii and Lactobacillus delbrueckii subsp. jakobsenii [3], [4]. L. lactis is the second subspecies used as a starter in the dairy industry along with L. bulgaricus within the L. delbrueckii species [3]. The in silico assembly of the ACA-DC 178 chromosome was validated against a NheI whole-genome optical map of the strain (Fig. 1). Our assembly presented 100% matching between the NheI restriction sites of the optical map and the relevant sites in our genome sequence in silico digested with the same enzyme. The genome was found to be 2,050,316 bp with a GC content of 49.6%. We were able to annotate a total of 2,112 genes, including 1,752 protein-coding genes, 239 putative pseudogenes, 94 tRNA and 27 rRNA genes (Fig. 2). Further analysis revealed that about 80% of the protein-coding genes (1,417 proteins) could be assigned to at least one Cluster of Orthologous Groups (COG) functional category. Most of these proteins (approximately 1/3) were distributed among three categories related to housekeeping processes, namely replication, recombination and repair (category L: 10.6%), translation, ribosomal structure and biogenesis (category J: 7.5%) and amino acid transport and metabolism (category E: 7.2%) (Table 1). Additional features of the ACA-DC 178 included 14 integrated genomic islands (GIs; Fig. 3) and a clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) system (Fig. 4). The GIs carry 159 genes some of which could be assigned to functions like exopolysaccharide biosynthesis, amino acid transport and restriction-modification. The CRISPR array was relatively long, consisting of 3,197 bp and 52 spacers. Detailed analysis of the spacers identified several segments of phage sequences, which have hosts belonging to the Lactobacillus genus.

Fig. 1

Validation of the L. lactis ACA-DC 178 genome assembly. Alignment of the in silico genome assembly of L. lactis ACA-DC 178 (bottom) against a NheI whole-genome optical map of the strain (top).

Fig. 2

Circular map of the L. lactis ACA-DC 178 genome. Each ring represents specific genomic features appearing from the periphery to the centre of the map: Forward CDSs (blue); Reverse CDSs (red); Pseudogenes (black); tRNA (green); rRNA (orange); %GC plot; GC skew.

Table 1

Distribution of L. lactis ACA-DC 178 proteins in COG categories.

	COG	Proteins	%	Description
Information storage and processing	J	132	7.5	Translation, ribosomal structure and biogenesis
	K	100	5.7	Transcription
	L	186	10.6	Replication, recombination and repair
Cellular processes and signaling	D	19	1.1	Cell cycle control, cell division, chromosome partitioning
	M	91	5.2	Cell wall/membrane biogenesis
	N	5	0.3	Cell motility
	O	47	2.7	Posttranslational modification, protein turnover, chaperones
	T	52	3.0	Signal transduction mechanisms
	U	19	1.1	Intracellular trafficking and secretion
	V	44	2.5	Defense mechanisms
Metabolism	C	46	2.6	Energy production and conversion
	E	127	7.2	Amino acid transport and metabolism
	F	67	3.8	Nucleotide transport and metabolism
	G	102	5.8	Carbohydrate transport and metabolism
	H	30	1.7	Coenzyme transport and metabolism
	I	32	1.8	Lipid transport and metabolism
	P	73	4.2	Inorganic ion transport and metabolism
	Q	3	0.2	Secondary metabolites biosynthesis, transport and catabolism
Poorly characterized	S	262	15.0	Function unknown
–	–	335	19.1	Not in COGs

Fig. 3

Circular map of the L. lactis ACA-DC 178 genome. Highlighted regions correspond to GIs. GIs are colored within the circular map according to the prediction method used: green, orange and blue were predicted by IslandPick, SIGI-HMM and IslandPath-DIMOB, respectively. The integrated GIs are presented on the periphery in red. The black line plot represents the GC content (%) of the genomic sequence.

Fig. 4

Graphical presentation of the CRISPR-Cas system of L. lactis ACA-DC 178.

Experimental design, materials, and methods

L. lactis ACA-DC 178 was grown overnight in MRS broth (Merck, Darmstadt, Germany) at 30 οC. DNA was extracted according to a previously published protocol [5]. The genome was sequenced at the Beijing Genomics Institute (BGI Co., Ltd, Hong Kong) using the Illumina HiSeq 2000 platform (Illumina, CA) employing paired-end libraries of 500 bp, 2,000 bp and 6,000 bp. The assembly of reads with SOAPdenovo v.2.04 [6] resulted in one circular chromosome that was verified against a NheI whole-genome optical map of the strain [7] produced at Microbion SRL (Verona, Italy). The alignment between the assembly and the optical map was performed with the MapSolver software (OpGen Technologies, Inc., Madison, WI). The ACA-DC 178 genome sequence was analyzed using Prodigal [8], MetaGeneAnnotator [9] and FGENESB [10] gene prediction programs. Genome annotation and prediction of rRNA and tRNA genes was performed with RAST v.2.0 [11] and putative pseudogenes were predicted with the GenePRIMP pipeline [12]. The results of the analysis were optimized with manual curation. COG annotations were computed using eggNOG-mapper based on eggNOG v.4.5 orthology database [13]. Further bioinformatic analysis was performed for the identification of GIs with IslandViewer 4 [14] and CRISPR with CRISPRFinder [15].

Specifications Table

Subject	Microbiology
Specific subject area	Genome analysis
Type of data	Table, Figure
How data were acquired	Genome sequencing: Illumina HiSeq 2000 (Illumina, CA), Denovo sequence assembly: SOAPdenovo v.2.04 software, MapSolver software (OpGen Technologies, Inc., Madison, WI), Bioinformatics approaches: Rapid Annotation using Subsystem Technology (RAST) v.2.0, Prodigal, MetaGeneAnnotator, FGENESB, GenePRIMP pipeline, eggNOG-mapper v.4.5, IslandViewer 4, CRISPRFinder
Data format	Raw sequence reads and genome assembly and annotation
Parameters for data collection	Genomic DNA from pure culture
Description of data collection	Purification of genomic DNA, genome sequencing, genome assembly and annotation
Data source location	Traditional Greek Kasseri Cheese, Athens, Greece
Data accessibility	Data are deposited in the respective databases and are publicly available. The annotated whole-genome sequence of L. lactis ACA-DC 178 is deposited at the European Nucleotide Archive (ENA) under the accession number LS991409 (https://www.ebi.ac.uk/ena/data/view/LS991409). Raw sequence reads are deposited in the Sequence Read Archive (SRA; SRR8866601-3; https://www.ncbi.nlm.nih.gov/sra/?term=SRR8866601; https://www.ncbi.nlm.nih.gov/sra/?term=SRR8866602; https://www.ncbi.nlm.nih.gov/sra/?term=SRR8866603).

Value of the Data• Lactobacillus delbrueckii subsp. lactis is an important bacterium used in cheese production. For this reason analysis of the genome sequence of strain ACA-DC 178 will provide valuable information for its adaptation in the milk environment and its technological properties. • Data presented here can be valuable for researchers involved in the field of genomic analysis of lactic acid bacteria and food fermentations. • Data can be used by researchers to perform comparative and functional genomics to further shed light in the evolution, biology and technological properties of the L. delbrueckii species. • Increasing the number of complete genome sequences within L. delbrueckii will further aid our understanding of this species.