Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group.

AIMS: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. METHODS AND RESULTS: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l-alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l-alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. CONCLUSIONS: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l-alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The greater ability of some strains to germinate in response to l-alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l-alanine.

Molecular cloning and characterization of apricot fruit polyphenol oxidase.

A reverse transcriptase-polymerase chain reaction experiment was done to synthesize a homologous polyphenol oxidase (PPO) probe from apricot (Prunus armeniaca var Bergeron) fruit. This probe was further used to isolate a full-length PPO cDNA, PA-PPO (accession no. AF020786), from an immature-green fruit cDNA library. PA-PPO is 2070 bp long and contains a single open reading frame encoding a PPO precursor peptide of 597 amino acids with a calculated molecular mass of 67.1 kD and an isoelectric point of 6.84. The mature protein has a predicted molecular mass of 56.2 kD and an isoelectric point of 5.84. PA-PPO belongs to a multigene family. The gene is highly expressed in young, immature-green fruit and is turned off early in the ripening process. The ratio of PPO protein to total proteins per fruit apparently remains stable regardless of the stage of development, whereas PPO specific activity peaks at the breaker stage. These results suggest that, in addition to a transcriptional control of PPO expression, other regulation factors such as translational and posttranslational controls also occur.

New spectrophotometric assay for polyphenol oxidase activity.

We describe a convenient and sensitive assay of polyphenol oxidase (PPO, EC 1.14.18.1) consisting of spectrophotometry at 300 nm based on the stoichiometric reaction of cysteine with o-quinones produced during the enzymatic oxidation of phenols. The adduct formed exhibited spectral properties different from those of the parent phenol. PPO activities extracted from apple, pear, and mushroom were assayed. The assay cannot be used with hydroxycinnamoyl derivatives since the cysteinyl adduct compounds exhibited spectral properties similar to those of their parent phenolic substrates. However, the cysteine-coupled method presents several advantages over the measurement of oxygen uptake by polarography or the direct estimation of o-quinone by spectrophotometry. The duration of the linear period was increased, allowing a better estimation of its value. The zone of proportionality between rates and enzyme quantities was enlarged. The difference in molar extinction coefficients between adduct and phenol at 300 nm ranged between 2000 and 2800 M-1.cm-1, i.e., two times higher than those of the corresponding o-quinones. Therefore, this assay improves the sensitivity of polyphenol oxidase detection over that of the direct spectrophotometric assay of quinone formation.