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Transposon Mutagenesis of the Obligate Intracellular Pathogen Rickettsia prowazekii. Aiping Qin, 2004.Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts . We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R . prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts . The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity . Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes . This R . prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsLP), and the entire construct was inserted into the Epicentre EZ::TN transposome system . A purified transposon containing rpsLP-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae . Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR . Multiple insertions into widely spaced areas of the R . prowazekii genome were identified . Three insertions were identified within gene coding sequences . Transposomes provide a mechanism for generating random insertional mutations in R . prowazekii, thereby identifying nonessential rickettsial genes . Erwinia chrysanthemi tolC Is Involved in Resistance to Antimicrobial Plant Chemicals and Is Essential for Phytopathogenesis  .Ravi D. Barabote, 2003.TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens . We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16 . The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue . The E . chrysanthemi gene was able to functionally complement the E . coli tolC gene with respect to its role in MDR efflux pumps . A tolC mutant of E . chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals . This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised . The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical . These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E . chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue . Microbial and Physiological Characterization of Weakly Amylolytic but Fast-Growing Lactic Acid Bacteria: a Functional Role in Supporting Microbial Diversity in Pozol, a Mexican Fermented Maize Beverage. G. Díaz-Ruiz, 2003.Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough . The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation . In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined . Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods . Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant . Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified . S . bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight]-1) and specific rate of amylase production (130.7 U g [cell dry weight]-1 h-1) . In contrast, it showed a high specific growth rate (0.94 h-1) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate-1) . These would confer on the strain a competitive advantage and are the possible reasons for its dominance . Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation . This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation . These results are the first step to understanding the importance of ALAB during pozol fermentation .
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