Genome analysis of two Lactobacillus curvatus strains that have emerged as dominant non-starter lactic acid bacteria in cheese

Abstract Text: Recent studies concerning cheese microbiology have revealed Lactobacillus curvatus becoming an increasingly common component of the non-starter lactic acid bacteria (NSLABs) population in aged Cheddar cheese. We recently sequenced the genome of two Lb. curvatus strains, WSU1, isolated from aged Cheddar cheese manufactured at Utah State University, and LFC-1, isolated from aged Cheddar cheese manufactured at the University of Wisconsin-Madison.  Whole-genome shotgun sequencing was performed on both isolates and assembled into draft genome sequences.  Preliminary genome annotation was performed using the RAST algorithm (rast.nmpdr.org). Initial genetic comparisons between the predicted coding sequences of the two strains showed similar genome content with strain WSU01 having 312 unique predicted proteins, and LFC-1 having 297 unique proteins using a 90% amino acid identity threshold. Both strains have genes encoding enzymes for cellobiose utilization, and the ability to ferment ribose and N-acetylglucosamine. Although both strains have genes for lactose utilization, API sugar panel results showed that only LFC-1 fermented lactose. Further analysis showed that LFC-1 also has genes that encode enzymes for maltose and trehalose fermentation, along with genes for citrate utilization. To understand why Lb. curvatus has become a dominant NSLAB in Cheddar cheese, genome sequences were analyzed to identify possible mechanisms. Both genomes contain genes for a putative sakacin-like bacteriocin and genes for a propanediol utilization pathway, which converts 1,2-propanediol to propanol and propionic acid. To test for propionate production, both strains were grown to carbohydrate exhaustion in MRS media (pH 5.2), after which 50 mM lactate was added. Cultures were incubated under strict anaerobic conditions for 22 d with samples taken after 0, 5, and 22 d and analyzed for propionate using GC-MS. Lactate concentrations were also measured using an enzymatic assay. Results revealed that both WSU1 and LFC-1 produce a 5-fold increase in propionate after 22 d. Previous studies on propionate production in other lactic acid bacteria show that lactate was converted to 1,2-propanediol, but lactate in both WSU1 and LFC-1 did not show a decrease in concentration during incubation. This observation opens the possibility that in Lb. curvatus, 1,2-propanediol is produced by another pathway and warrants further investigation since high levels of propionic acid have been found in aged Cheddar cheese where Lb. curvatus is a dominant NSLAB. 

Keywords: NSLABs, cheese, genomes