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The Lactococcal Abortive Phage Infection System AbiP Prevents both Phage DNA Replication and Temporal Transcription Switch. Susana Domingues, 2004.We describe here a new lactococcal abortive phage infection system, designated AbiP . AbiP is effective against some lactococcal phages of one prevalent group, 936, but not against phages from the other two groups (c6A and P335) . It was identified in the Lactococcus lactis subsp . cremoris strain IL420, on the native plasmid pIL2614 . AbiP is encoded by a single gene, expressed in an operon with a second gene . In this work, abiP is shown to affect both the replication and transcription of phage DNA . In AbiP+ cells, phage DNA replication is arrested approximately 10 min after infection . Levels of middle and late phage transcripts are lower in AbiP+ than in AbiP- cells, probably due to the smaller amount of phage DNA . By contrast, early phage transcripts are more abundant in AbiP+ than in AbiP- cells, suggesting that the switch-off, which occurs 15 min after infection in AbiP- cells, is prevented in AbiP+ cells . Characterization of the Double-Partitioning Modules of R27: Correlating Plasmid Stability with Plasmid Localization. Trevor D. Lawley, 2003.Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2 . Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase) . Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant . Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form . Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1- and Par2-) under different growth conditions in live Escherichia coli cells . Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells . Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E . coli . Both R27 Par- variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm . Those cells that did contain R27 Par- with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27 . Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm . Fungus-Elicited Metabolites from Plants as an Enriched Source for New Leishmanicidal Agents: Antifungal Phenyl-Phenalenone Phytoalexins from the Banana Plant (Musa acuminata) Target Mitochondria of Leishmania donovani Promastigotes. Juan Román Luque-Ortega, 2004.Two antifungal phenyl-phenalenone phytoalexins isolated from the banana plant (Musa acuminata) elicited with the fungus Fusarium oxysporum, together with a methoxy derivative of one of them and two epoxide precursors of their chemical synthesis, were tested for leishmanicidal activity on Leishmania donovani promastigotes and L . infantum amastigotes . Drugs inhibited proliferation of both forms of the parasite with a 50% lethal concentration range between 10.3 and 68.7 µg/ml . Their lethal mechanism was found linked to the respiratory chain by a systematic approach, including electron microscopy, measurement of the oxygen consumption rate on digitonin-permeabilized promastigotes, and enzymatic assays on a mitochondrial enriched fraction . Whereas the whole set of compounds inhibited the activity of fumarate reductase in the mitochondrial fraction (50% effective concentration [EC50] between 33.3 and 78.8 µg/ml) and on purified enzyme (EC50 = 53.3 to 115 µg/ml), inhibition for succinate dehydrogenase was only observed for the two phytoalexins with the highest leishmanicidal activity: anigorufone and its natural analogue 2-methoxy-9-phenyl-phenalen-1-one (EC50 = 33.5 and 59.6 µg/ml, respectively) . These results provided a new structural motif, phenyl-phenalenone, as a new lead for leishmanicidal activity, and support the use of plant extracts enriched in antifungal phytoalexins, synthesized under fungal challenge, as a more rational and effective strategy to screen for new plant leishmanicidal drugs . Transcriptional Analysis of Biofilm Formation Processes in the Anaerobic, Hyperthermophilic Bacterium Thermotoga maritima. Marybeth A. Pysz, 2004.Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C . A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations . Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T . maritima genome . Among the previously annotated genes in the T . maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis) . Most notably, T . maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones . These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells . Significant up-regulation of several ß-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat . The reasons for increased ß-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides . In addition to revealing insights into the phenotype of sessile T . maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments . One Repeat of the Cell Wall Binding Domain Is Sufficient for Anchoring the Lactobacillus acidophilus Surface Layer Protein. Egbert Smit, 2002.The N-terminal repeat (SAC1) of the S-protein of Lactobacillus acidophilus bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not . Treatment of CWFs with hydrofluoric acid, but not phenol, prevented binding . Apparently, SAC1 is necessary and sufficient for cell wall binding . Our data suggest that SAC anchors the S-protein to a cell wall teichoic acid . ccfA, the Genetic Determinant for the cCF10 Peptide Pheromone in Enterococcus faecalis OG1RF. Michelle H. Antiporta, 2002.The nosocomial pathogen Enterococcus faecalis has a unique pheromone-inducible conjugative mating system . Conjugative transfer of the E . faecalis plasmid pCF10 is specifically induced by the cCF10 peptide pheromone (LVTLVFV) . Genomic sequence information has recently allowed the identification of putative structural genes coding for the various enterococcal pheromones (D . B . Clewell et al., Mol . Microbiol . 35:246-247, 2000) . The cCF10 pheromone sequence LVTLVFV was found within an open reading frame designated ccfA, encoding a putative lipoprotein precursor . Several other pheromone sequences were found in similar locations within other predicted lipoproteins . CcfA shows significant sequence relatedness to the Escherichia coli protein YidC, an inner membrane protein translocase, as well as to a large number of homologs identified in gram-positive and in gram-negative bacteria . Analysis of the deduced CcfA amino acid sequence suggested that mature cCF10 peptide could be formed from the proteolytic degradation of its signal peptide . Expression of the cloned ccfA gene with an inducible expression vector dramatically increased cCF10 production by E . faecalis and also resulted in cCF10 production by Lactococcus lactis, a non-pheromone producer . Site-directed mutagenesis of the ccfA sequence encoding the cCF10 peptide confirmed that ccfA was a functional genetic determinant for cCF10 . Involvement of Calcium/Calmodulin Signaling in Cercosporin Toxin Biosynthesis by Cercospora nicotianae. Kuang-Ren Chung, 2003.Cercosporin is a non-host-selective, perylenequinone toxin produced by many phytopathogenic Cercospora species . The involvement of Ca2+/calmodulin (CaM) signaling in cercosporin biosynthesis was investigated by using pharmacological inhibitors . The results suggest that maintaining endogenous Ca2+ homeostasis is required for cercosporin biosynthesis in Cercospora nicotianae . The addition of excess Ca2+ to the medium slightly increased fungal growth but resulted in a reduction in cercosporin production . The addition of Ca2+ chelators [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] also reduced cercosporin production . Ca2+ channel blockers exhibited a strong inhibition of cercosporin production only at higher concentrations (>2 mM) . Cercosporin production was reduced greatly by Ca2+ ionophores (A23187 and ionomycin) and internal Ca2+ blocker [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester] . Phospholipase C inhibitors (lithium, U73122, and neomycin) led to a concentration-dependent inhibition of cercosporin biosynthesis . Furthermore, the addition of CaM inhibitors (compound 48/80, trifluoperazine, W-7, and chlorpromazine) also markedly reduced cercosporin production . In contrast to W-7, W-5, with less specificity for CaM, led to only minor inhibition of cercosporin production . The inhibitory effects of Ca2+/CaM inhibitors were partially or completely reversed by the addition of external Ca2+ . As assessed with Fluo-3/AM (a fluorescent Ca2+ indicator), the Ca2+ content in the cytoplasm decreased significantly when fungal cultures were grown in a medium containing Ca2+/CaM antagonists, confirming the specificity of those Ca2+/CaM antagonists in C . nicotianae . Taken together, the results suggest that Ca2+/CaM signal transduction may play a pivotal role in cercosporin biosynthesis in C . nicotianae .
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