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Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp . To Identify Sources of Fecal Pollution. X. Bonjoch, 2004.Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution . The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences . Only B . adolescentis and B . dentium were found exclusively in human sewage . A multiplex PCR approach with strain-specific primers was developed . The method showed a sensitivity threshold of 10 cells/ml . This new molecular method could provide useful information for the characterization of fecal pollution sources . Differential Effects of Mutations in tonB1 on Intrinsic Multidrug Resistance and Iron Acquisition in Pseudomonas aeruginosa. Qixun Zhao, 2002.Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa . Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only . Accumulation of S-Adenosyl-L-Methionine Enhances Production of Actinorhodin but Inhibits Sporulation in Streptomyces lividans TK23. Dong-Jin Kim, 2003.S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine . Despite extensive research with many organisms, its role in Streptomyces sp . remains unclear . In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis . The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein . The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media . Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters . In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell . From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp. Control of rep Gene Expression in Plasmid pGA1 from Corynebacterium glutamicum. Tatiana Venkova-Canova, 2003.The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth group of rolling-circle-replicating plasmids . A determinant, which negatively controls pGA1 replication, was localized in the leader region of the rep gene coding for the initiator of plasmid replication . This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48 . A promoter and a single transcriptional start site were found in the rep leader region in orientation opposite to the rep gene . These results suggest that a small countertranscribed RNA (ctRNA) (ca . 89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed . Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria . Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number . A region between the promoters Prep and P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low activity of the rep promoter even when promoter P-ctRNA was deleted . Thus, the sequence within the rep leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1 rep gene .
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