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Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of Pseudomonas aeruginosa. Jocelyn K. Middlemiss, 2004.The integral inner membrane resistance-nodulation-division [RND] components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems . To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a deltamexB strain . Of 100 mutants that expressed wild-typelevels of MexB and showed increased susceptibility to one ormore of carbenicillin, chloramphenicol, nalidixic acid, andnovobiocin, the mexB genes of a representative 46 were sequenced,and 19 unique single mutations were identified . While the majorityof mutations occurred within the large periplasmic loops betweentransmembrane segment 1 [TMS-1] and TMS-2 and between TMS-7and TMS-8 of MexB, mutations were seen in the TMSs and in otherperiplasmic as well as cytoplasmic loops . By threading the MexBamino acid sequence through the crystal structure of the homologousRND transporter from Escherichia coli, AcrB, a three-dimensionalmodel of a MexB trimer was obtained and the mutations were mappedto it . Unexpectedly, most mutations mapped to regions of MexBpredicted to be involved in trimerization or interaction withMexA rather than to regions expected to contribute to substraterecognition . Intragenic second-site suppressor mutations thatrestored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction . As theG220S mutation likely impacted trimerization, it appears thateither proper assembly of the MexB trimer is necessary for OprMinteraction or OprM association with an unstable MexB trimermight stabilize it, thereby restoring activity. Response of Bacillus subtilis to Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside. Charles M. Moore, 2004.We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression . In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival . DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B . subtilis to NO and SNP . Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress . Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur . In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II) . Aerobically, NO gas induced hmp, the  B
general stress regulon, and, to a lesser extent, the Fur and PerR
regulons . Surprisingly, NO gas induced the
B
regulon via the energy branch of the
B
regulatory cascade while induction by SNP was mediated by the
environmental stress branch . This emphasizes that NO and SNP elicit
genetically distinct stress responses .
LuxS-Based Signaling in Streptococcus gordonii: Autoinducer 2 Controls Carbohydrate Metabolism and Biofilm Formation with Porphyromonas gingivalis. Roderick McNab, 2003.Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms . In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm . S . gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi . An isogenic mutant of S . gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces . In contrast, the mutant strain failed to induce bioluminescence in V . harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis . Complementation of the luxS mutation in S . gordonii restored normal biofilm formation with the luxS-deficient P . gingivalis . Differential display PCR demonstrated that the inactivation of S . gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-ß-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase . However, S . gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S . gordonii and P . gingivalis, was unaffected by inactivation of luxS . The results suggest that S . gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism . Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P . gingivalis and S . gordonii . Identification and Characterization of Phytoplasmal Genes, Employing a Novel Method of Isolating Phytoplasmal Genomic DNA. Sharon Melamed, 2003.Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes . To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant . Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA . Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized . We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins . Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed . Factors Influencing Survival of Legionella pneumophila Serotype 1 in Hot Spring Water and Tap Water. Akira Ohno, 2003.The factors involved in the survival of Legionella pneumophila in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity . L . pneumophila could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42°C, except for one microcosm with a pH of <2.0 . Lower temperatures supported survival without loss of cultivability . The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45°C . The optimal range of pH for survival was between 6.0 and 8 . The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt . The cultivability of organisms in the post-exponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase . In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase . Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected . Resuscitation by the addition of Acanthamoeba castellanii to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population . Our results suggest that L . pneumophila cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas .
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