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Regulation of IcsP, the Outer Membrane Protease of the Shigella Actin Tail Assembly Protein IcsA, by Virulence Plasmid Regulators VirF and VirB. Helen J. Wing, 2004.The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread . Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB . In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter . Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF . In contrast, when VirF was induced in the absence of VirB, VirF had variable effects . The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB . We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP . Leucines 193 and 194 at the N-Terminal Domain of the XylS Protein, the Positive Transcriptional Regulator of the TOL meta-Cleavage Pathway, Are Involved in Dimerization. Raquel Ruíz, 2003.Members of the AraC/XylS family of transcriptional regulators are usually organized in two domains: a conserved domain made up of 100 amino acids and frequently located at the C-terminal end, involved in DNA binding; and an N-terminal nonconserved domain involved in signal recognition, as is the case for regulators involved in the control of carbon metabolism (R . Tobes and J . L . Ramos, Nucleic Acids Res . 30:318-321, 2002) . The XylS protein, which is extremely insoluble, controls expression of the meta-cleavage pathway for alkylbenzoate metabolism . We fused the N-terminal end of XylS to the maltose-binding protein (MBP) in vitro and found in glutaraldehyde cross-linking assays that the protein dimerized . Experiments with a chimeric N-terminal XylS linked to a 'LexA protein showed that the dimer was stabilized in the presence of alkylbenzoates . Sequence alignments with AraC and UreR allowed us to identify three residues, Leu193, Leu194, and Ile205, as potentially being involved in dimerization . Site-directed mutagenesis of XylS in which each of the above residues was replaced with Ala revealed that Leu193 and Leu194 were critical for activity and that a chimera in which LexA was linked to the N terminus of XylSLeu193Ala or XylSLeu194Ala was not functional . Dimerization of the chimeras MBP-N-XylSLeu193Ala and MBP-N-XylSLeu194Ala was not observed in cross-linking assays with glutaraldehyde . Quorum-Sensing Signals and Quorum-Sensing Genes in Burkholderia vietnamiensis. Barbara-Ann Conway, 2002.Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many Proteobacteria including a clinical isolate of Burkholderia cepacia . The B . cepacia isolate produces low levels of octanoyl-HSL . We have examined an environmental isolate of Burkholderia vietnamiensis . This isolate produced several acyl-HSLs . The most abundant species was decanoyl-HSL . Decanoyl-HSL in B . vietnamiensis cultures reached concentrations in excess of 20 µM . We isolated a B . vietnamiensis DNA fragment containing a gene for the synthesis of decanoyl-HSL (bviI) and an open reading frame that codes for a putative signal receptor (bviR) . A B . vietnamiensis bviI mutant did not produce detectable levels of decanoyl-HSL . Mutational Analysis of Cell Wall Biosynthesis in Mycobacterium avium. Jean-Pierre Laurent, 2003.The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance . To identify genes involved in cell wall biosynthesis, "transposome" insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red . Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M . avium genome . Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichia coli Strains. Z. Ignatova, 2003.Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E . coli expression hosts . Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production . Rate constants for both intracellular proteolytic breakdown (kd) and transport (kt) were used as quantitative tools for selection of the appropriate host system and cultivation medium . The production of mature active PA was increased up to 10-fold when the protease-deficient strain E . coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants kd and kt . The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E . coli host strains . Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production . In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein) . The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture . Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank. E. Nsabimana, 2003.The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen . The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum . We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival . In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S . Ki  idayová, J . Microbiol . Methods 22:185-192, 1995), we found that a holding temperature of -30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate . Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium . They were not significantly modified after a period of 1 year in liquid nitrogen . Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals . These results have made it possible to set up a bank of cryopreserved rumen protozoa .
Induction and Resuscitation of Viable but Nonculturable Salmonella enterica Serovar Typhimurium DT104  .A. R. Gupte, 2003.Salmonella enterica serovar Typhimurium DT104 11601was tested for its ability to maintain viability in minimal, chemically defined solutions . Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms (  235 days) at 5°C . Organisms maintained at a higher temperature (21°C) showed continuous, equivalent CFU per milliliter (106) up to 400 days after inoculation . Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity . Temperature upshift (56°C ± 0.5, 15 s) of the nonculturable organisms (5°C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation . Interestingly, S . enterica serovar Typhimurium DT104 from the microcosms at either 5°C (1 to 200 days) or 21°C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA . Furthermore, cells from 21°C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells . It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans . The fact that S . enterica serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that S . enterica serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions .
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