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Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in Mycobacterium tuberculosis as It Goes into a Dormancy-Like State in Culture. Jaiyanth Daniel, 2004.Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy . The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops . Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known . We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids . Little is known about the biosynthesis of TG in M . tuberculosis . We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes . We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O2 and also by NO treatment, which is known to induce dormancy-associated genes . The gene (Rv3130c) that shows the highest TGS activity when expressed in E . coli shows the highest induction by hypoxia and NO treatment . Biochemical evidence shows that TG synthesis and accumulation occur under both conditions . We conclude that TG may be a form of energy storage for use during long-term dormancy . Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis . Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection. Gary J. Vora, 2004.DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities . However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification . Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection . To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based,  29 DNA polymerase-based, and multiplex PCR . The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples . Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach . However, the use of Klenow-plus-Klenow and
29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR . In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR . The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA . The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources .
Metabolic Signals That Lead to Control of CBB Gene Expression in Rhodobacter capsulatus. Mary A. Tichi, 2002.Various mutant strains were used to examine the regulation and metabolic control of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus . Previously, a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient strain (strain SBI/II) was found to show enhanced levels of cbbI and cbbII promoter activities during photoheterotrophic growth in the presence of dimethyl sulfoxide . With this strain as the starting point, additional mutations were made in genes encoding phosphoribulokinase and transketolase and in the gene encoding the LysR-type transcriptional activator, CbbRII . These strains revealed that a product generated by phosphoribulokinase was involved in control of CbbR-mediated cbb gene expression in SBI/II . Additionally, heterologous expression experiments indicated that Rhodobacter sphaeroides CbbR responded to the same metabolic signal in R . capsulatus SBI/II and mutant strain backgrounds . The Ascorbate Transporter of Escherichia coli. Zhongge Zhang, 2003.The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane  -helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively . We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of L-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro . The transporter exhibits an apparent Km for L-ascorbate of 9 µM and is highly specific . The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr . The yjfR gene product is essential for L-ascorbate utilization and probably encodes a cytoplasmic L-ascorbate 6-phosphate lactonase . We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to L-ascorbate via the phosphoryl transfer pathway:
PEP
 enzyme I-P
HPr-P
IIA-
IIB-
 L-ascorbate-6-P .
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