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Extracellular Proteolytic Activity Plays a Central Role in Swarming Motility in Bacillus subtilis. Mariah Bindel Connelly, 2004.Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming . A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface . As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria . While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria . To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility . Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm . While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function . These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms . Population Pharmacokinetics of Indinavir in Patients Infected with Human Immunodeficiency Virus. Chantal Csajka, 2004.Indinavir is currently used at a fixed dose of 800 mg either three times a day or twice a day in combination with 100 mg of ritonavir . Dosage individualization based on plasma concentration monitoring might, however, be indicated . This study aimed to assess the pharmacokinetic profile of indinavir in patients infected with human immunodeficiency virus to characterize interpatient and intrapatient variability and to build up a Bayesian approach for dosage adaptation . A population analysis was performed with the NONMEM computer program with 569 plasma samples from a cohort of 239 unselected patients receiving indinavir . A one-compartment model with first-order absorption was adapted, and the influences of clinical characteristics on oral clearance (CL) and distribution volume (V) were examined . Predicted average drug exposure and trough and peak concentrations were derived for each patient and correlated with efficacy and toxicity markers . The population estimates of CL were 32.4 liters/h for female and 42.0 liters/h for male patients; oral V was 65.7 liters; and the rate constant of absorption (Ka) was 1.0 h–1 . CL decreased by 63% with ritonavir intake and was moderately correlated to body weight . Both interpatient variability, best assigned to oral CL (coefficient of variation [CV], 39%) and Ka (CV, 67%), and intrapatient variability were large (CV, 41%; standard deviation, 670 µg/liter) . In conclusion, initial indinavir dosage should be decided according to ritonavir intake and sex, prior to plasma concentration measurements . The high interpatient pharmacokinetic variability represents an argument for therapeutic drug monitoring . Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae). Melinda R. Duplessis, 2004.The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria . The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis . Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions . In contrast, the symbionts of C . orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking . Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) . In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate . However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions . Autolysis and Autoaggregation in Pseudomonas aeruginosa Colony Morphology Mutants. David A. D'Argenio, 2002.Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants . One set of mutants formed wrinkled colonies of autoaggregating cells . Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation . WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria . The second set of distinctive insertion mutants formed colonies that lysed at their center . Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing . Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS . The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1 . The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P . aeruginosa between survival of the many and persistence of the few . Analysis of bvgR Expression in Bordetella pertussis. Tod J. Merkel, 2003.Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease . The expression and regulation of these virulence factors are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein . It is hypothesized that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator, which upon modification by BvgS binds to specific promoters and activates transcription . An additional class of genes is repressed by the products of the bvg locus . The repression of these genes is dependent upon the third gene, bvgR . Expression of bvgR is dependent upon the function of BvgA and BvgS . This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR . We undertook an analysis of the transcriptional activation of bvgR expression . We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension . We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA . Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses . A model of BvgA binding to the bvgR promoter is presented . Novel Approach to the Improvement of Biphenyl and Polychlorinated Biphenyl Degradation Activity: Promoter Implantation by Homologous Recombination. Yoshiyuki Ohtsubo, 2003.To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon (bphEGF[orf4]A1A2A3CD[orf1]A4R) of Pseudomonas sp . strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity . A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity . By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs . This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation . An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization. Raju Sekar, 2003.We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes . The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes . In particular, the abundances of aquatic Actinobacteria were significantly underestimated . We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples . Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss . Incubations with high concentrations of lysozyme (10 mg ml-1) followed by achromopeptidase (60 U ml-1) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples . Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes . For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%) . Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%) . Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton . Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats. Pedro F. B. Brandão, 2003.A molecular screening approach was developed in order to amplify the genomic region that codes for the  - and ß-subunits of the nitrile hydratase (NHase) enzyme in rhodococci . Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis . A hydratase PCR product was also obtained from R . erythropolis DSM 43066T, which did not grow on nitriles . Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R . erythropolis strains used as positive controls . PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R . erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin . Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases . The phylogenetic analysis and deduced amino acid sequences suggested that the novel R . erythropolis enzymes belonged to the iron-type NHase family . Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes . A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R . erythropolis . Our findings indicate that the NHase and amidase genes present in geographically distinct R . erythropolis strains are not globally mixed .
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