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The Left End of IS2: a Compromise between Transpositional Activity and an Essential Promoter Function That Regulates the Transposition Pathway. Leslie A. Lewis, 2004.Cut-and-paste (simple insertion) and replicative transposition pathways are the two classical paradigms by which transposable elements are mobilized . A novel variation of cut and paste, a two-step transposition cycle, has recently been proposed for insertion sequences of the IS3 family . In IS2 this variation involves the formation of a circular, putative transposition intermediate (the minicircle) in the first step . Two aspects of the minicircle may involve its proposed role in the second step (integration into the target) . The first is the presence of a highly reactive junction formed by the two abutted ends of the element . The second is the assembly at the minicircle junction of a strong hybrid promoter which generates higher levels of transposase . In this report we show that IS2 possesses a highly reactive minicircle junction at which a strong promoter is assembled and that the promoter is needed for the efficient completion of the pathway . We show that the sequence diversions which characterize the imperfect inverted repeats or ends of this element have evolved specifically to permit the formation and optimal function of this promoter . While these sequence diversions eliminate catalytic activity of the left end (IRL) in the linear element, sufficient sequence information essential for catalysis is retained by the IRL in the context of the minicircle junction . These data confirm that the minicircle is an essential intermediate in the two-step transposition pathway of IS2 . Comparison of Compositions and Metabolic Activities of Fecal Microbiotas in Young Adults and in Antibiotic-Treated and Non-Antibiotic-Treated Elderly Subjects. Emma J. Woodmansey, 2004.The colonic microbiota mediates many cellular and molecular events in the host that are important to health . These processes can be affected in the elderly, because in some individuals, the composition and metabolic activities of the microbiota change with age . Detailed characterizations of the major groups of fecal bacteria in healthy young adults, in healthy elderly people, and in hospitalized elderly patients receiving antibiotics were made in this study, together with measurements of their metabolic activities, by analysis of fecal organic acid and ammonia concentrations . The results showed that total anaerobe numbers remained relatively constant in old people; however, individual bacterial genera changed markedly with age . Reductions in numbers of bacteroides and bifidobacteria in both elderly groups were accompanied by reduced species diversity . Bifidobacterial populations in particular showed marked variations in the dominant species, with Bifidobacterium angulatum and Bifidobacterium adolescentis being frequently isolated from the elderly and Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium boum, and Bifidobacterium infantis being detected only from the healthy young volunteers . Reductions in amylolytic activities of bacterial isolates in healthy elderly subjects and reduced short-chain fatty acid concentrations supported these findings, since bifidobacteria and bacteroides are important saccharolytic groups in the colon . Conversely, higher numbers of proteolytic bacteria were observed with feces samples from the antibiotic-treated elderly group, which were also associated with increased proteolytic species diversity (fusobacteria, clostridia, and propionibacteria) . Other differences in the intestinal ecosystem in elderly subjects were observed, with alterations in the dominant clostridial species in combination with greater numbers of facultative anaerobes . Nonenzymatic Turnover of an Erwinia carotovora Quorum-Sensing Signaling Molecule. Joseph T. Byers, 2002.The production of virulence factors and carbapenem antibiotic in the phytopathogen Erwinia carotovora is under the control of quorum sensing . The quorum-sensing signaling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), accumulates in log-phase culture supernatants of E . carotovora but diminishes in concentration during the stationary phase . In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur . Rather, it was caused by degradation of the molecule . The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing . The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to  8.5 . OHHL became unstable over a narrow pH range (pH 7 to 8) . Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30°C) by buffering the samples at pH 6.8 . These results may provide a rationale for the observation that an early response of plants which are under attack by Erwinia is to activate a proton pump which alkalizes the site of infection to a pH of >8.2 .
Identification of a Regulated Alkaline Phosphatase, a Cell Surface-Associated Lipoprotein, in Mycobacterium smegmatis. Jordan Kriakov, 2003.Although alkaline phosphatases are common in a wide variety of bacteria, there has been no prior evidence for alkaline phosphatases in Mycobacterium smegmatis . Here we report that transposon insertions in the pst operon, encoding homologues of an inorganic phosphate transporter, leads to constitutive expression of a protein with alkaline phosphatase activity . DNA sequence analysis revealed that M . smegmatis does indeed have a phoA gene that shows high homology to other phoA genes . The M . smegmatis phoA gene was shown to be induced by phosphate starvation and thus negatively regulated by the pst operon . Interestingly, the putative M . smegmatis PhoA has a hydrophobic N-terminal domain which resembles a lipoprotein signal sequence . The M . smegmatis PhoA was demonstrated to be an exported protein associated with the cell surface . Furthermore, immunoprecipitation of PhoA from [14C]acetate-labeled M . smegmatis cell lysates demonstrated that this phosphatase is a lipoprotein . Biotin Limitation in Sinorhizobium meliloti Strain 1021 Alters Transcription and Translation. Elke B. Heinz, 2003.Most Sinorhizobium meliloti strains lack several key genes involved in microbial biotin biosynthesis, and it is assumed that this may be a special adaptation which allows the microbe to down-regulate metabolic activities in the absence of a host plant . To further explore this hypothesis, we employed two different strategies . (i) Searches of the S . meliloti genome database in combination with the construction of nine different gusA reporter fusions identified three genes involved in a biotin starvation response in this microbe . A gene coding for a protein-methyl carboxyl transferase (pcm) exhibited 13.6-fold-higher transcription under biotin-limiting conditions than cells grown in the presence of 40 nM biotin . Consistent with this observation, biotin-limiting conditions resulted in a significantly decreased survival of pcm mutant cells compared to parental cells or cells grown in the presence of 40 nM biotin . Further studies indicated that the autoinducer synthase gene, sinI, was transcribed at a 4.5-fold-higher level in early stationary phase in biotin-starved cells than in biotin-supplemented cells . Lastly, we observed that open reading frame smc02283, which codes for a putative copper resistance protein (CopC), was 21-fold down-regulated in response to biotin starvation . (ii) In a second approach, proteome analysis identified 10 proteins which were significantly down-regulated under the biotin-limiting conditions . Among the proteins identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry were the  subunit of the RNA polymerase and the 50S ribosomal protein L7/L12 (L8) subunit, indicating that biotin-limiting conditions generally affect transcription and translation in S . meliloti .
Diffusional Properties of Methanogenic Granular Sludge: 1H NMR Characterization. Piet N. L. Lens, 2003.The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging . NMR spectroscopy measurements were performed at 22°C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons) . Self-diffusion coefficients of H2O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water . Interestingly, self-diffusion coefficients of H2O were independent of the aggregate size for the size fractions investigated . Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55°C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10°C) temperatures . Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90°C for 30 min) modified the diffusional transport up to 20% . In contrast, deactivation of aggregates by HgCl2 did not affect the H2O self-diffusion coefficient in aggregates . Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors .
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