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Molecular Characterization of the eis Promoter of Mycobacterium tuberculosis. Esteban A. Roberts, 2004.To further understand Mycobacterium tuberculosis pathogenesis, the regulation of potential virulence genes needs to be investigated . The eis gene of M . tuberculosis H37Rv enhances the intracellular survival of Mycobacterium smegmatis, which does not contain eis, within macrophages (J . Wei, J . L . Dahl, J . W . Moulder, E . A . Roberts, P . O'Gaora, D . B . Young, and R . L . Friedman, J . Bacteriol . 182:377-384, 2000) . Experiments were done to characterize the eis promoter in M . smegmatis and M . tuberculosis H37Ra . The putative –10 and –35 regions matched the Escherichia coli  70
consensus 67 and 83%, respectively, making it a group A/SigA-like
mycobacterial promoter . Expression of site-directed variants of the
core promoter region, determined by flow cytometry using gfp
as a reporter, showed that the putative –10 region is essential for
eis expression . In addition, site-directed alteration of the
eis promoter to the consensus E . coli
70
promoter elements increased gfp transcription to levels
similar to that driven by the heat shock promoter, phsp60, of
Mycobacterium bovis BCG . Upstream promoter deletion analysis
showed that a 200- and 412-bp region of the promoter was necessary
for maximum expression of gfp in M . smegmatis and M .
tuberculosis H37Ra, respectively . Random mutagenesis of the
412-bp eis promoter, using a catechol 2,3-dioxygenase screen
and activity assay, defined nucleotides upstream of the core promoter
region that are essential to eis expression in both M .
smegmatis and M . tuberculosis H37Ra, including a region
homologous to a DinR cis element .
Purification and Polar Localization of Pneumococcal LytB, a Putative Endo-ß-N-Acetylglucosaminidase: the Chain-Dispersing Murein Hydrolase. Blanca De Las Rivas, 2002.The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in Escherichia coli . LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-ß-N-acetylglucosaminidase of Streptococcus pneumoniae . LytB can remove a maximum of only 25% of the radioactivity from [3H]choline-labeled pneumococcal cell walls in in vitro assays . Inactivation of the lytB gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence . Longer chains were formed when the lytB mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of lytA, which codes for the major autolysin . Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus . Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively . In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced . On the other hand, the use of a translational fusion between the gene (gfp) coding for the green fluorescent protein (GFP) and lytB supports the notion that LytB accumulates in the cell poles of either the wild-type R6, lytB mutants, or ethanolamine-containing cells (EA cells) . The GFP-LytB fusion protein was also able to unchain the lytB mutants but not the EA cells . In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length . These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells . NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes. Jian-Lin Yu, 2002.Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates . The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2 . Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His . In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment . Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K+-valinomycin having little effect . Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin . In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition . NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture . NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil . These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter . Thermal Inactivation of Susceptible and Multiantimicrobial-Resistant Salmonella Strains Grown in the Absence or Presence of Glucose. R. T. Bacon, 2003.The heat resistance of susceptible and multiantimicrobial-resistant Salmonella strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE-G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE-G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61°C . Cultures were heated in sterile 0.1% buffered peptone water (50 µl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath . Decimal reduction times (D values) were calculated from survival curves having r2 values of >0.90 as a means of comparing thermal tolerance among variables . D59°C values increased (P < 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE-G, TSBYE, and TSBYE+G cultures, respectively . D61°C values of antimicrobial-susceptible Salmonella strains increased (P < 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while D61°C values of multiantimicrobial-resistant Salmonella strains did not differ (P > 0.05) between TSBYE-G and TSBYE+G cultures . When averaged across glucose levels and temperatures, there were no differences (P > 0.05) between the D values of susceptible and multiantimicrobial-resistant inocula . Collectively, D values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for Salmonella strains inactivated at 55, 57, 59, and 61°C, respectively . zD values were 1.20, 1.48, and 1.49°C for Salmonella strains grown in TSBYE+G, TSBYE, and TSBYE-G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol . Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress .
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