Gluconate metabolism and gas production by Paucilactobacillus wasatchensis WDC04.

Paucilactobacillus wasatchensis, a nonstarter lactic acid bacteria, can cause late gas production and splits and cracks in aging cheese when it metabolizes 6-carbon substrates, particularly galactose, to a 5-carbon sugar, resulting in the release of CO2. Previous studies have not explained late gas production in aging cheese when no galactose is present. Based on the genome sequence of Pa. wasatchensis WDC04, genes for potential metabolic pathways were mapped using knowledgebase predictive biology software. This metabolic modeling predicted Pa. wasatchensis WDC04 could metabolize gluconate. Gluconate contains 6 carbons, and Pa. wasatchensis WDC04 contains genes to convert it to 6-P-gluconate and then to ribulose-5-P by using 6-phosphogluconate dehydrogenase in a decarboxylating step, producing CO2 during its metabolism. The goal of this study was to determine if sodium gluconate, often added to cheese to reduce calcium lactate crystal formation, could be metabolized by Pa. wasatchensis WDC04, resulting in gas production. Carbohydrate-restricted DeMan, Rogosa, and Sharpe broth was mixed with varying ratios of ribose, sodium gluconate, or d-galactose (total added substrate content of 1% wt/vol). Oxyrase (Oxyrase Inc.; 1.8% vol/vol) was also used to mimic the anaerobic environment of cheese aging in selected tubes. Tubes were inoculated with a 4-d culture of Pa. wasatchensis WDCO4, and results were recorded over 8 d. When inoculated into carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing only sodium gluconate as the added substrate, Pa. wasatchensis WDC04 grew, confirming gluconate utilization. Of the 10 ratios used, Pa. wasatchensis WDC04 produced gas in 6 scenarios, with the most gas production resulting from the ratio of 100% sodium gluconate with no added ribose or galactose. It was confirmed that obligately heterofermentative nonstarter lactobacilli such as Pa. wasatchensis WDC04 can utilize sodium gluconate to produce CO2 gas. Addition of sodium gluconate to cheese thus becomes another risk factor for unwanted gas production and formation of slits and cracks.

Growth and survival characteristics of Paucilactobacillus wasatchensis WDCO4.

Understanding characteristics that permit survival and growth of Paucilactobacillus wasatchensis as part of the nonstarter microbiota of cheese is important for minimizing unwanted gas formation in cheese that can cause downgrading because of slits and cracks. The ability of Plb. wasatchensis WDC04 to survive pasteurization was studied by inoculating raw milk with 108 cfu/mL and measuring survival after processing through a high-temperature, short-time pasteurizer. Extent and rate of growth of Plb. wasatchensis WDC04 as a function of pH, salt concentration, and presence of various organic acids were studied using 48-well microplates in an automated spectrophotometer measuring optical density at 600 nm. Better growth in the 1-mL wells was obtained when a micro-anaerobic environment (similar to that which occurs in cheese) was created by enzymically removing the oxygen. Faster growth occurred around neutral pH (pH 6 to 8) than at pH 5 (cheese pH), whereas only marginal growth occurred at pH 4. Adding sodium chloride retarded growth of Plb. wasatchensis WDC04, but slow growth occurred even at salt concentrations up to 6%. At salt-in-moisture (S/M) concentrations found in cheese, the rate of growth at 3.5% S/M >4.5% S/M >5.5% S/M. Thus, low salt level in cheese is a risk factor for Plb. wasatchensis growth during cheese storage and unwanted slits and cracks. Some of the organic acids tested (propionic, formic, and citric) tended to suppress growth of Plb. wasatchensis WDC04 more than would be expected from their effect on pH. No survival of Plb. wasatchensis WDC04 after pasteurization was observed with the reduction in numbers being 8 logs or more. Even subpasteurization heating at 69°C for 15 s was sufficient to inactivate Plb. wasatchensis WDC04, so its presence as part of the nonstarter microbiota of cheese should be considered as a postpasteurization environmental contamination.

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup.

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.