Lactobacillus helveticus MTCC 5463 was isolated from a vaginal swab from a healthy adult female. The strain exhibited potential probiotic properties, with their beneficial role in the gastrointestinal tract and their ability to reduce cholesterol and stimulate immunity. We sequenced the whole genome and compared it with the published genome sequence of Lactobacillus helveticus DPC4571.Lactobacillus helveticus is present in fermented foods and is also used as a probiotic (4,10,12). Researchers have evaluated the effect of L. helveticus against diseases such as cancer and intestinal inflammation. Milk fermented with L. helveticus R389 delayed breast tumor growth by decreasing interleukin-6 (IL-6) and increasing IL-10 in serum, mammary glands, and tumor-infiltrating immune cells (5). Lactobacillus helveticus strains are normally isolated from milk products and intestinal microflora (6,14). The L. helveticus MTCC 5463 strain was originally isolated from the vaginal tract of a healthy adult female in India at Anand Agricultural University (9). The L. helveticus MTCC 5463 strain, earlier known as Lactobacillus acidophilus V3 (based on biochemical characteristics) was able to grow in the presence of 0.3% sodium taurocholate, deconjugate bile acids, and reduce cholesterol in vitro (1). The strain exhibited significant antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, and Escherichia coli (7). The strain produced extracellular polysaccharide and was able to adhere to cells of the human carcinoma cell line HT29. A hypocholesterolemic effect of L. helveticus MTCC 5463 was reported in human subjects with different cholesterol levels (2). The strain has also shown positive immunomodulating effects in a chick model (13).The whole-genome sequencing of L. helveticus MTCC 5463 was performed using GS-FLX Titanium reagents (11). The data generated from the genomic library contained 119,569 reads, and assembly generated a 1,911,350-bp single chromosome. The genome annotation and comparative analysis of the genome were done with the published genome of strain DPC4571 (3). The genomic sequence of L. helveticus MTCC 5463 was somewhat smaller than those of L. helveticus DPC4571 (2.08 Mb) and L. helveticus H10 (2.14 Mb). In total, 2,046 coding sequence (CDS) regions and 71 RNA genes were reported. Of the 71 RNA genes, 59 coded for tRNA, 8 for rRNA, and 4 for 5S RNA. The CDS regions were slightly fewer than in L. helveticus H10 (2,049) and L. helveticus DPC4571 (2,238). Metabolic reconstruction subsystems were assembled to create a metabolic reaction network for L. helveticus MTCC 5463 and L. helveticus DPC4571.The subsystem analysis revealed a common subsystem structure between L. helveticus MTCC 5463 and L. helveticus DPC4571 for seven subsystems, viz., photosynthesis, iron acquisition and metabolism, motility and chemotaxis, secondary metabolism, stress response, nitrogen metabolism, and dormancy and sporulation. The considerable variation observed in the ...