Genotypic and Phenotypic Characterization of Highly Alkaline-Resistant Carnobacterium maltaromaticum V-Type ATPase from the Dairy Product Based on Comparative Genomics

Abstract

Although Carnobacterium maltaromaticum derived from dairy products has been used as a lactic acid bacterium industrially, several studies have reported potential pathogenicity and disease outbreaks. Because strains derived from diseased fish and dairy products are considered potentially virulent and beneficial, respectively, their genotypic and phenotypic characteristics have attracted considerable attention. A genome-wide comparison of 30 genome sequences (13, 3, and 14 strains from diseased aquatic animals, dairy products, and processed food, respectively) was carried out. Additionally, one dairy and two nondairy strains were incubated in nutrient-rich (diluted liquid media) and nutrient-deficient environments (PBS) at pH 10 to compare their alkaline resistance in accordance with different nutritional environments by measuring their optical density and viable bacterial cell counts. Interestingly, only dairy strains carried 11 shared accessory genes, and 8 genes were strongly involved in the V-type ATPase gene cluster. Given that V-type ATPase contributes to resistance to alkaline pH and salts using proton motive force generated via sodium translocation across the membrane, C. maltaromaticum with a V-type ATPase might use nutrients in food under high pH. Indeed, the dairy strain carrying the V-type ATPase exhibited the highest alkaline resistance only in the nutrient-rich environment with significant upregulation of V-type ATPase expression. These results suggest that the gene cluster of V-type ATPase and increased alkaline resistance of dairy strains facilitate adaptation in the long-term ripening of alkaline dairy products.

Keywords: V-type ATPase; alkaline resistance; bioinformatics; food quality; genomics; lactic acid bacteria.

Figure 1

Three-dimensional principal component analysis (PCA) of strains derived from dairy products, diseased fish, and processed food based on the profiling of KEGG Ontology (ko) genes (A). Clustering of 30 strains of Carnobacterium maltaromaticum isolated under different conditions (B). The accessory genes and ko_id that shared only dairy strains among 30 strains of C. maltaromaticum (C). The serial array of sulfate permease (SulP), transposase (Tn), and the components of V-type ATPase (ATPVA, ATPVB, ATPVC, ATPVD, ATPVE, ATPVF, ATPVI, and ATPVK) in the dairy strains of chromosomal DNA (D).

Figure 2

PCR products (ATPVA- and ATPVI-specific primers) using 18ISCm, ATCC35586, and DSM20342 genomic DNA (A). The results of in silico PCR for all 30 strains. The results of in silico PCR for all 30 strains isolated from dairy products, diseased fish, and processed food. + denotes positive amplification and the expected amplification size is indicated in the parentheses. - denotes no amplification in in silico PCR (B).

Figure 3

Relative OD value at 8, 24, 48, and 96 hpi in 1/10 TSB (pH 10; (A)) and pH 10 PBS (pH 10; (B)), respectively. Viable bacterial count at 24 and 48 hpi in 1/10 TSB (pH 10: (C)) and pH 10 PBS (pH 10; (D)), respectively. Different letters indicate statistically significant differences determined by Duncan’s multiple range test under the same sampling time points (p < 0.05).

Figure 4

Relative V-type ATPase Subunit A (ATPVA) gene expression in DSM20342 strain under PBS and 1/10 TSB at pH 10. The dots in the box plot indicate the fold change of each sample compared to hpi0. Different letters indicate statistically significant differences determined via Duncan’s multiple range test among all groups (p < 0.05).

Figure 5

Schematic diagram about the mode of action for V-type ATPase, which only exists in dairy C. maltaromaticum under a highly alkaline environment. ∆ψ indicated the electrical potential in intra- and extra-cellular areas.