Computational protocol: Complete Genome Sequence of Streptococcus thermophilus Strain B59671, Which Naturally Produces the Broad-Spectrum Bacteriocin Thermophilin 110

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Protocol publication

[…] Streptococcus thermophilus is a nonpathogenic lactic acid bacterium commonly isolated from bovine mammary tissue and raw milk, and it is used as a starter culture for the production of yogurt and cheese. Some S. thermophilus strains have been investigated for their production of broad-spectrum antimicrobial peptides called bacteriocins. Several strains naturally produce bacteriocins (), while others require a synthetic 30-mer quorum-sensing peptide, BlpC30, to induce production (). The chromosomal locus encoding BlpC30-induced bacteriocins was designated the bacteriocin-like peptide (blp) gene cluster when characterized in strains LMD-9, LMG18311, and CNRZ1066 (). The blp gene cluster varied substantially within these strains, and only LMD-9 was induced to express an active bacteriocin (, ).Here, we report the complete genome sequence of S. thermophilus strain B59671, which naturally produces a broad-spectrum bacteriocin, thermophilin 110, encoded within the blp gene cluster (, ). Thermophilin 110 was reported to inhibit the growth of Listeria monocytogenes (), Streptococcus mutans, Streptococcus pyogenes, and Propionibacterium acnes (). In addition, similar to S. thermophilus strains Y2 () and APC151 (), strain B59671 expresses glutamate decarboxylase and is capable of converting glutamate to gamma-aminobutyric acid (GABA) (), a potent neurotransmitter reported to have analgesic, antihypertensive, and antidiabetic activities ().Following growth in tryptone-yeast extract-lactose medium, cells were pretreated with mutanolysin, and genomic DNA was isolated using the DNeasy blood and tissue kit (Qiagen), according to the manufacturer’s protocol. Genome sequencing was performed by the Genomics Core Facility, Clinical and Translational Research Institute, Drexel College of Medicine (Philadelphia, PA) with a Pacific Biosciences RSII system (PacBio, Menlo Park, CA). Single-molecule real-time (SMRT) sequencing was performed with P6/C4 PacBio chemistry. De novo assembly was achieved with HGAP assembler version 2.3, yielding a single contig supported by a mean coverage of 542-fold (). The genome was circularized by permutation to begin at the dnaA gene, and terminal duplications were removed using Circlator version 1.0.2. The genome was resequenced with RS_Modification_and_Motif_Anlysis version 2.3, and Prokka version 1.11 () and Taxator version 1.2 () were used for analysis. The fully assembled genome consisted of a single chromosome of 1,821,173 bp.Genome annotation was obtained by the NCBI Prokaryotic Genome Annotation Pipeline (Bethesda, MD) and revealed a total of 1,936 genes, 67 tRNAs, and 6 rRNAs (16S and 23S). In addition, there were 196 pseudogenes identified, which agreed with previous reports showing that approximately 10% of the genes in S. thermophilus are nonfunctional (). The Clone Manager software (Sci-Ed Software, NC) was used to identify homologs of blpA and blpX, which flank the blp gene cluster (). In B59671, this locus contained 10,067 bp, which was 2,501 and 2,172 bp less than the corresponding loci in strains LMD-9 and LMG18311, respectively (). The nucleotide sequence from B59671 matched the sequences in LMD-9 and LMG18311 by 76 and 79%, respectively. Further studies will aim to fully characterize the blp gene cluster in B59671. […]

Pipeline specifications

Software tools HGAP, Circlator, Prokka, PGAP
Application De novo sequencing analysis
Organisms Streptococcus thermophilus
Chemicals gamma-Aminobutyric Acid, Nucleotides, Lactic Acid