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Null Mutation of HvrA Compensates for Loss of an Essential relA/spoT-Like Gene in Rhodobacter capsulatus. Shinji Masuda, 2004.We report that a single relA/spoT-like gene exists on the Rhodobacter capsulatus chromosome, and its mutational loss is lethal . This gene could be mutated only under a mutational background of a null mutation in the nucleoid protein HvrA . This result suggests that there may be a direct link between HvrA-regulated promoters and the ppGpp-related stringent response . The Antimalarial Potential of 4-Quinolinecarbinolamines May Be Limited due to Neurotoxicity and Cross-Resistance in Mefloquine-Resistant Plasmodium falciparum Strains. Geoffrey S. Dow, 2004.The clinical potential of mefloquine has been compromised by reports of adverse neurological effects . A series of 4-quinolinecarbinolamines were compared in terms of neurotoxicity and antimalarial activity in an attempt to identify replacement drugs . Neurotoxicity (MTT [thiazolyl blue reduction] assay) was assessed by exposure of cultured embryonic rat neurons to graded concentrations of the drugs for 20 min . The 50% inhibitory concentration (IC50) of mefloquine was 25 µM, while those of the analogs were 19 to 200 µM . The relative (to mefloquine) therapeutic indices of the analogs were determined after using the tritiated hypoxanthine assay for assessment of the antimalarial activity of the analogs against mefloquine-sensitive (W2) and -resistant (D6 and TM91C235) Plasmodium falciparum strains . Five analogs, WR157801, WR073892, WR007930, WR007333, and WR226253, were less neurotoxic than mefloquine and exhibited higher relative therapeutic indices (RTIs) against TM91C235 (2.9 to 12.2) . Conventional quinoline antimalarials were generally less neurotoxic (IC50s of 400, 600, and 900 for amodiaquine, chloroquine, and quinine) or had higher RTIs (e.g., 30 for halofantrine against TM91C235) . The neurotoxicity data for the 4-quinolinecarbinolamines were used to develop a three-dimensional (3D), function-based pharmacophore . The crucial molecular features correlated with neurotoxicity were a hydrogen bond acceptor (lipid) function, an aliphatic hydrophobic function, and a ring aromatic function specifically distributed in the 3D surface of the molecule . Mapping of the 3D structures of a series of structurally diverse quinolines to the pharmacophore allowed accurate qualitative predictions of neurotoxicity (or not) to be made . Extension of this in silico screening approach may aid in the identification of less-neurotoxic quinoline analogs . Identification of Sigma Factor  B-Controlled Genes and Their Impact on Acid Stress, High Hydrostatic Pressure, and Freeze Survival in Listeria monocytogenes EGD-e.Henrike H. Wemekamp-Kamphuis, 2004.The gene encoding the alternative sigma factor B in Listeria monocytogenes is induced upon exposure of cells to several stresses . In this study, we investigated the impact of a sigB null mutation on the survival of L . monocytogenes EGD-e at low pH, during high-hydrostatic-pressure treatment, and during freezing . The survival of
 sigB mutant exponential-phase cells at pH 2.5 was 10,000-fold lower than the survival of EGD-e wild-type cells . Moreover, the
sigB mutant failed to show an acid tolerance response . Upon preexposure for 1 h to pH 4.5, the survival at pH 2.5 was 100,000-fold lower for the
sigB mutant than for the wild type . The glutamate decarboxylase (GAD) acid resistance system is important in survival and adaptation of L . monocytogenes in acidic conditions . The
B dependence of the gad genes (gadA, gadB, gadC, gadD, and gadE) was analyzed in silico . Putative
B-dependent promoter sites were found upstream of the gadCB operon (encoding a glutamate/ -aminobutyrate antiporter and a glutamate decarboxylase, respectively) and the lmo2434 gene (gadD, encoding a putative glutamate decarboxylase) . Reverse transcriptase PCR revealed that expression of the gadCB operon and expression of gadD are indeed
B dependent . In addition, a proteomics approach was used to analyze the protein expression profiles upon acid exposure . Although the GAD proteins were not recovered, nine proteins accumulated in the wild type but not in the
sigB strain . These proteins included Pfk, GalE, ClpP, and Lmo1580 . Exposure to pH 4.5, in order to preload cells with active
B and consequently with
B-dependent general stress proteins, also provided considerable protection against high-hydrostatic-pressure treatment and freezing . The combined data argue that the expression of
B-dependent genes provides L . monocytogenes with nonspecific multiple-stress resistance that may be relevant for survival in the natural environment as well as during food processing .
Biofilm Formation and Acyl Homoserine Lactone Production in the Burkholderia cepacia Complex. Barbara-Ann D. Conway, 2002.Acyl homoserine lactone (acyl-HSL)-mediated gene regulation has been shown to influence biofilm formation in one Burkholderia cepacia cystic fibrosis isolate, but it is not known whether this relationship is a consistent feature of the several genomic species that make up the B . cepacia complex (BCC) . We screened strains belonging to genomovars I to V of the BCC for biofilm formation on an abiotic surface and for acyl-HSL synthesis . We determined that organisms from each of these genomovars were capable of biofilm formation . Similarly, acyl-HSL was synthesized by organisms from each of genomovars I to V, with most isolates producing octanoyl-HSL in greatest abundance . When biofilms were grown in Luria broth, acyl-HSL synthesis and biofilm formation appeared to be associated, but these phenotypes were independent when the biofilms were grown in basal salts containing citrate . Genomovar V strains synthesized the greatest quantities of acyl-HSL, and genomovar II and III-A strains elaborated the most abundant biofilms . Quorum sensing may play a role in BCC pathogenesis, but it may not regulate biofilm formation under all growth conditions . TetR Family Member PsrA Directly Binds the Pseudomonas rpoS and psrA Promoters. Milan Kojic, 2002.We have previously described a Pseudomonas gene, psrA, which enhances transcription of the rpoS sigma factor gene at stationary phase . We present molecular data which demonstrate that in Pseudomonas putida PsrA binds specifically to the rpoS and psrA promoters in DNA regions having similar palindromic sequences, C/GAAAC N2-4 GTTTG/C, where N is any nucleotide . The position of the initiation of transcription was determined for both promoters, and PsrA binds from positions -59 to -35 in the rpoS promoter and from -18 to +20 in the psrA promoter with respect to the +1 transcription site . Expression studies with a psrA-lacZ transcriptional fusion in wild-type and psrA::Tn5 knockout mutants revealed that psrA was under additional control in response to growth phase . A model for the role of PsrA in the regulation of rpoS and psrA is presented . The Ti Plasmid of Agrobacterium tumefaciens Harbors an attM-Paralogous Gene, aiiB, Also Encoding N-Acyl Homoserine Lactonase Activity. A. Carlier, 2003.The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of Bacillus . When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host . In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta .
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