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Drug Susceptibility and Genetic Evaluation of Plasmodium falciparum Isolates Obtained in Four Distinct Geographical Regions of Kenya. Abigael Mbaisi, 2004.The drug resistance profiles of Plasmodium falciparum isolated from four regions in Kenya were analyzed for drug resistance profiles . We observed variability in resistance to a broad range of antimalarial drugs across Kenya as determined from in vitro drug susceptibility screening and genotyping analysis . Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR. Jan Kostal, 2004.The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic . Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth . Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite . Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression . Conversely, no significant improvement in Cd2+ or Zn2+ accumulation was observed, validating the specificity of ArsR . The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S . Environmental Protection Agency limit of 10 ppb . These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water . The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway. Alexander D. Kim, 2002.The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and L-alanine (L-Ala) . Here, we describe the purification and characterization of recombinant AEPT . pH rate profiles (log Vm and log Vm/Km versus pH) revealed a pH optimum of 8.5 . At pH 8.5, Keq is equal to 0.5 and the kcat values of the forward and reverse reactions are 7 and 9 s-1, respectively . The Km for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for L-Ala it is 1.4 ± 0.03 mM . Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation . The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily . Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences . Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis . Collaborative Regulation of Escherichia coli Glutamate-Dependent Acid Resistance by Two AraC-Like Regulators, GadX and GadW (YhiW). Zhuo Ma, 2002.An important feature of Escherichia coli pathogenesis is an ability to withstand extremely acidic environments of pH 2 or lower . This acid resistance property contributes to the low infectious dose of pathogenic E . coli species . One very efficient E . coli acid resistance system encompasses two isoforms of glutamate decarboxylase (gadA and gadB) and a putative glutamate:  -amino butyric acid (GABA) antiporter (gadC) . The system is subject to complex controls that vary with growth media, growth phase, and growth pH . Previous work has revealed that the system is controlled by two sigma factors, two negative regulators (cyclic AMP receptor protein [CRP] and H-NS), and an AraC-like regulator called GadX . Earlier evidence suggested that the GadX protein acts both as a positive and negative regulator of the gadA and gadBC genes depending on environmental conditions . New data clarify this finding, revealing a collaborative regulation between GadX and another AraC-like regulator called GadW (previously YhiW) . GadX and GadW are DNA binding proteins that form homodimers in vivo and are 42% homologous to each other . GadX activates expression of gadA and gadBC at any pH, while GadW inhibits GadX-dependent activation . Regulation of gadA and gadBC by either regulator requires an upstream, 20-bp GAD box sequence . Northern blot analysis further indicates that GadW represses expression of gadX . The results suggest a control circuit whereby GadW interacts with both the gadA and gadX promoters . GadW clearly represses gadX and, in situations where GadX is missing, activates gadA and gadBC. GadX, however, activates only gadA and gadBC expression . CRP also represses gadX expression . It does this primarily by repressing production of sigma S, the sigma factor responsible for gadX expression . In fact, the acid induction of gadA and gadBC observed when rich-medium cultures enter stationary phase corresponds to the acid induction of sigma S production . These complex control circuits impose tight rein over expression of the gadA and gadBC system yet provide flexibility for inducing acid resistance under many conditions that presage acid stress .
Growth Dynamics of Salmonella enterica Strains on Alfalfa Sprouts and in Waste Seed Irrigation Water. Michael B. Howard, 2003.Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis . The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process . To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds . An S . enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process . Germinating alfalfa seeds supported the multiplication of S . enterica cells prior to the emergence of the root radicle at 72 h . Thereafter, much lower rates of multiplication were apparent . The ability of S . enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain . Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools . Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds . The S . enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source . S . enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination . The ability to detect S . enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations . Statistical Approaches for Estimating Actinobacterial Diversity in Marine Sediments. James E. M. Stach, 2003.Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m . Clones were placed into operational taxonomic unit (OTU) groups with  99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth . The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth . Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively . Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries . FST and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages . It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T . Palys, L . K . Nakamura, and F . M . Cohan, Int . J . Syst . Bacteriol . 47:1145-1156, 1997) . Mechanisms responsible for diversity and their implications are discussed .
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