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New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis. D. Llull, 2004.Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn2+ high-affinity uptake and regulation . A PZn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic ß-galactosidase . Nuclease and ß-galactosidase activities of L . lactis MG1363 cells expressing either uspnuc or lacLM under the control of PZn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn2+, availability in the environment . Our results demonstrate that PZn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn2+ . The efficiency of the PZn zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either PZn zitR or PnisA nisRK control . lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the PZn zitR system . An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection . PZn zitR proved to be a powerful expression system for L . lactis, as it is tightly controlled by the zinc concentration in the medium . Effect of Translational Signals on mRNA Decay in Bacillus subtilis. Josh S. Sharp, 2003.A 254-nucleotide model mRNA, designated  ermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis .
ermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure . Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability . Data for the decay of
ermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation—even the formation of the first peptide bond—was not important for stability . The half-life of an untranslated 3.2-kb
ermC-lacZ fusion RNA was similar to that of a translated
ermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability . The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5'-end-dependent activity, possibly a 5'-binding endonuclease, which is required for the initiation of mRNA decay . This model is supported by the finding that increasing the distance from the 5' end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life . These results underscore the importance of the 5' end to mRNA stability in B . subtilis .
Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing. Cristina Tun-Garrido, 2003.Rhizobium etli CFN42 contains six plasmids . Only one of them, p42a, is self-conjugative at high frequency . This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym) . To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated . Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids . Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family . Mutagenesis of these genes confirmed their requirement for p42a transfer . We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer . Although R . etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C8-homoserine lactone encoded by the traI gene described here, was involved in transfer . Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer . Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer . Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C8-homoserine lactone synthesized by TraI . The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI . Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a . Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis. Hiroyuki Inoue, 2003.The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT . Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized . The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P . chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads . Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I . Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants . F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively . The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production . The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine . On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu2+ and Sn4+, respectively . These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states . Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products. Gianluca Bleve, 2003.Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds . Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage . From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells . The optimized RT-PCR assay was able to detect 10 CFU of fungi ml-1 in pure culture and 103 and 102 CFU ml-1 in artificially contaminated yogurts and pasteurized fruit-derived products, respectively . Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi . The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities .
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