Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.     

Comparison of European and US biological indicators for ethylene oxide sterilization

Summary Biological indicators (BIs) are used to monitor ethylene oxide (EO) gas sterilization processes for medical devices. Several European and United States BIs for EO sterilization were evaluated for resistance according to both United States Pharmacopeia (USP) XXI and United Kingdom's (UK) tests for D-values. US BIs areB. subtilis var. niger spores on paper strips or disc carriers while European BIs use aluminum strips, quartz sand, or cotton yarn. Numerous BIs per run and runs per lot, as well as 2–3 different lots of BIs from each manufacturer, were examined. Both British and US BIs met their respective label claims for rates of inactivation when tested against British and USP EO test parameters, respectively. However, Danish BIs, on cotton yarn or quartz sand, were not inactivated following USP specifications during the exposure dwell times tested (600 mg L^–1 EO, 54°C, 60% RH, 0–110 min). The Danish BIs will require further testing in order for us to determine if theirB. subtilis spores are unusually resistant to EO or if the spore carrier substrates protect the spores from the sterilizing gas. In conclusion, the British and American BIs for EO sterilization are equivalent in resistance despite differences in carrier substrate, recovery conditions, calculation methods for D-values, and the labeled sterilization conditions for use.     

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.     

Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.     

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72–98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-β family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-β family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF). Received: 21 August 2000 / Accepted: 9 March 2001