Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO₂ supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.     

Genetic analysis of the anti-mutagenic effect of genistein in Escherichia coli

Genistein, the main isoflavone in soy, has received considerable attention for its potential anti-carcinogenic properties. In a previous report, we investigated the possible role of genistein in anti-mutagenesis, using an Escherichia coli reversion assay system. Genistein reduced ENU-induced mutagenesis in a dose-dependent manner and the reduction of mutation frequency was differential among several categories of mutation. Most notable was a loss of transversion mutations, which require SOS functions. In this report, we further investigated the anti-mutagenic effect of genistein using a genetic approach. E. coli strains having alterations in genes involved in SOS-mutagenesis were examined, as were strains having defects in proteins that might serve as potential targets for genistein. The results showed that ENU-induced mutations produced in recA730 and lexA(Def) strains, both expressing a constitutive SOS response, were reduced by genistein to a lesser extent than in the wild-type strain. The effect of genistein was not entirely abolished, however. ENU mutagenesis in a umuC derivative, which reflects predominantly transition mutations, was unaffected by genistein. ENU-induced mutations in strains having defects in topA, gyrA, typA or uspA were not different than the wild-type, suggesting that these gene products were not involved in genistein's anti-mutagenic effect. In addition, we determined the distribution of genistein in various cellular fractions using HPLC. These studies revealed that genistein could be recovered from E. coli cells grown on agar media containing genistein; the intracellular concentration was similar to that in the agar plates. Further, most of the genistein recovered was associated with proteins in the cytosolic fraction and little partitioned in the membrane fraction. In vitro studies showed that genistein could be precipitated from a protein (BSA) containing solution. Finally, we examined the effect of genistein on formation of the RecA filament on ssDNA in vitro and observed an inhibition at high concentrations of genistein. In total, these results suggested that genistein may reduce SOS-dependent mutagenesis by reducing the interaction of RecA protein with ssDNA. As a consequence, genistein could cause a reduction in (1) the overall SOS response (confirmed using β-galactosidase assays) and (2) trans-lesion DNA synthesis by DNA polymerase V.     

Salmonella phage ST64B encodes a member of the SseK/NleB effector family

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.