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Inactivations of rsbU and sarA by IS256 Represent Novel Mechanisms of Biofilm Phenotypic Variation in Staphylococcus epidermidis. Kevin M. Conlon, 2004.Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation . Reversible transposition of IS256 into icaADBC or downregulation of icaADBCexpression are two important mechanisms of biofilm phenotypicvariation . Interestingly, the presence of IS256 was generallyassociated with a more rapid rate of phenotypic variation, suggestingthat IS256 insertions outside the ica locus may affect ica transcription.Consistent with this, we identified variants with diminishedica expression, which were associated with IS256 insertionsin the  B
activator rsbU or sarA . Biofilm development and ica
expression were activated only by ethanol and not NaCl in rsbU::IS256
insertion variants, which were present in
 11%
of all variants.
B
activity was impaired in rsbU::IS256 variants, as evidenced
by reduced expression of the
B-regulated
genes asp23, csb9,and rsbV . Moreover,
expression of sarA, which is
B
regulated,and SarA-regulated RNAIII were also suppressed . A
biofilm-formingphenotype was restored to rsbU::IS256
variants only after repeatedpassage and was not associated with IS256
excision from rsbU.Only one sarA::IS256
insertion mutant was identified among 43biofilm-negative variants .
Both NaCl and ethanol-activated icaexpression in this sarA::IS256
variant, but only ethanol increasedbiofilm development . Unlike
rsbU::IS256 variants, reversionof the sarA::IS256
variant to a biofilm-positive phenotype wasaccompanied by precise
excision of IS256 from sarA and restorationof normal
ica expression . These data identify new roles forIS256
in ica and biofilm phenotypic variation and demonstratethe
capacity of this element to influence the global regulationof
transcription in S . epidermidis.
A Series of Diaryltriazines and Diarylpyrimidines Are Highly Potent Nonnucleoside Reverse Transcriptase Inhibitors with Possible Applications as Microbicides. Yven Van Herrewege, 2004.An in vitro model of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells, representing the primary targets of sexual human immunodeficiency virus (HIV) transmission, was used to evaluate the antiviral and immune suppressive activity of new classes of nonnucleoside reverse transcriptase inhibitors, diaryltriazines (DATAs) and diarylpyrimidines (DAPYs), compared to the reference compounds UC-781 and PMPA . Antiviral activity (as reflected by the 50% effective concentration [EC50]) was determined by treating HIV-infected MO-DC/CD4+-T-cell cocultures with a dose range of a compound during 14 days, followed by analysis of supernatants in HIV p24 antigen enzyme-linked immunosorbent assay . A limited, 24-h treatment evaluated the compounds as microbicides . Viral rescue was evaluated in a PCR by monitoring proviral DNA in secondary cultures with phytohemagglutinin-interleukin-2 blasts . We determined 50% immunosuppressive concentrations in mixed leukocyte cultures of MO-DC and allogeneic T cells, with compound either continuously present or present only during the first 24 h . The EC50 values of DATA and DAPY compounds ranged from 0.05 to 3 nM compared to 50 nM for UC-781 and 89 nM for PMPA . When evaluated in the "microbicide" setting, the most potent compounds completely blocked HIV infection at 10 to 100 nM . The immunosuppressive concentrations were well above the EC50, resulting in favorable therapeutic indices for all compounds tested . The DATA and DAPY compounds described here are more potent than earlier reverse transcriptase inhibitors and show favorable pharmacological profiles in vitro . They could strengthen the antiretroviral armamentarium and might be useful as microbicides . Multivariate Analyses of Burkholderia Species in Soil: Effect of Crop and Land Use History. Joana Falcão Salles, 2004.The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications . Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia . In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles . The diversity of Burkholderia spp . in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA) . CCA ordination plots showed that previous land use was the main factor affecting the composition of the Burkholderia community . Although most variation in the Burkholderia community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed . Plant species affected Burkholderia community structure to a lesser extent than did land use history . Similarities were observed between Burkholderia populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand . Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation . The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with Burkholderia species that are commonly isolated from soil, in particular Burkholderia glathei, B . caledonica, B . hospita, and B . caribiensis . Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli K-12. Sabine Enz, 2003.Transcription of the fecABCDE ferric citrate transport genes of Escherichia coli K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm . FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor . In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR237-317 fragment interacts with the N-terminal FecA1-79 fragment . In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis . They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins . The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium . Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E) . One FecR mutant, FecR (D138E, V197A), induced fecA promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA . The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the fec transport genes and identified sites in FecR and FecA that are important for signal transduction . Conversion of 2-Fluoromuconate to cis-Dienelactone by Purified Enzymes of Rhodococcus opacus 1cp. Inna P. Solyanikova, 2003.The present study describes the 19F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol . The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica . Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified . ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone . Especially the cycloisomerization of 2-fluoromuconate is a new observation . ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone . The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R . opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol .
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