Whole-Genome Sequence of the Cheese Isolate Streptococcus macedonicus 679

Streptococcus macedonicus has been identified as part of the fermenting flora of dairy foods around the world, and it presents some common traits to the well-established dairy starter Streptococcus thermophilus (1, 2). However, S. macedonicus belongs to the Streptococcus bovis/Streptococcus equinus complex, which includes species with a zoonotic potential that have been associated, among other conditions, with endocarditis, meningitis, and colon cancer (2, 3). Here, we present the genome sequence of S. macedonicus 679 isolated from French uncooked semihard cheese made with cow milk (4) that may facilitate the assessment of its safety when ingested with foods.

The genome of S. macedonicus 679 was sequenced by mate-pair Illumina sequencing (Mr DNA, Shallowater, TX). The library was prepared using the Nextera mate-pair sample prep kit (Illumina, San Diego, CA). Genome DNA (gDNA) was isolated and quantified. Subsequently, the sample underwent fragmentation, strand displacement, circularization, shearing, streptavidin purification, end repair, adenylation, and adapter ligation. The ligated adapters were utilized during a limited-cycle (10 cycles) PCR. Following the library preparation, the average library size was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The library was sequenced by using the 600-cycle version 3 reagent kit (Illumina) in MiSeq (Illumina). Reads were assembled using SPAdes (5) against the published sequences of the chromosome and plasmid pSMA198 of S. macedonicus ACA-DC 198 as templates (6, 7). This reference-driven assembly resulted in two chromosomal contigs (contig_1, 79,307 bp; contig_2, 2,014,050 bp), as well as one plasmid contig (contig_3, 14,059 bp).

We employed an annotation transfer strategy using PANNOTATOR (8) and as reference the annotation of the S. macedonicus ACA-DC 198 genome. PANNOTATOR annotations were further enriched with RAST (9). The annotation transfer resulted in a total of 2,246 coding sequences (CDSs) in S. macedonicus 679 (84 in contig_1, 2,143 in contig_2, and 19 in contig_3). Among these CDSs, 75.1%, 7.7%, and 17.2% were 100%, 100 to 70%, and <70% identical to genes of S. macedonicus ACA-DC 198, respectively. We then inspected manually all annotation transfers of pseudogenes from strain ACA-DC 198 to strain 679. We identified 155 potential pseudogenes. This increased percentage of pseudogenes in the genome of S. macedonicus 679 is in agreement with previous observations for S. macedonicus ACA-DC 198, suggesting a genome decay evolution pattern that may be related to adaptation of the species to the rich in nutrients environment of milk (2, 6). Even though the majority of pseudogenes were common between the two strains, we could identify some that were present in one but not the other strain. This clearly indicates that genome decay took place early in the evolution of the species, but strain-specific selective pressures also generated distinct pseudogenes. The S. macedonicus 679 genome needs further investigation to better establish in silico its safety, its technological potential, and its adaptation traits to the dairy environment.