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Biofilm Development and Cell Death in the Marine Bacterium Pseudoalteromonas tunicata. Anne Mai-Prochnow, 2004.The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment . As part of our studies on the ecology of P . tunicata and its interaction with marine surfaces, we examined the ability of P . tunicata to form biofilms under continuous culture conditions within the laboratory . P . tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels . Remarkably, we observed a repeatable pattern of cell death during biofilm development of P . tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J . S . Webb et al., J . Bacteriol . 185:4585-4595, 2003) . Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures . A subpopulation of viable cells was always observed within the regions of killing in the biofilm . Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum . A novel 190-kDa autotoxic protein produced by P . tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment . A  alpP mutant derivative of P . tunicata was generated, and this mutant did not show cell death during biofilm development . We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P . tunicata and subsequent dispersal of surviving cells within the marine environment .
Mutational Scanning and Affinity Cleavage Analysis of UhpA-Binding Sites in the Escherichia coli uhpT Promoter. Igor N. Olekhnovich, 2002.UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate . UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from -80 to -50 and the weak-binding (W) region from -50 to -32 . Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits . Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding . Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription . These W region variant promoters were strongly stimulated by CAP . Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro . Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region . None of these substitutions eliminated promoter function . The orientation of UhpA-binding sites was assessed by the affinity cleavage method . The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA . Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions . These results are consistent with the binding of two dimers of UhpA . Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp . It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets . Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis. Matija Hedl, 2002.Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis . However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E . I . Wilding et al., J . Bacteriol . 182:4319-4327, 2000) . Enzymes of the mevalonate pathway are thus potential targets for drug intervention . Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase . Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae . The mvaE gene was cloned from E . faecalis and was expressed with an N-terminal His tag in Escherichia coli . The gene product was then purified by nickel affinity chromatography . As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions . Temperature optima,  Ha and Km values, and pH optima were determined for both activities . Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism . CoA acted as an inhibitor competitive with acetyl-CoA . A millimolar Ki for a statin drug confirmed that E . faecalis HMG-CoA reductase is a class II enzyme . The oxidoreductant was NADP(H) . A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde . Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756 . The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme .
Novel Methylotrophy Genes of Methylobacterium extorquens AM1 Identified by using Transposon Mutagenesis Including a Putative Dihydromethanopterin Reductase. Christopher J. Marx, 2003.Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen . One of these genes encodes a product having identity with dihydrofolate reductase (DHFR) . This mutant has a C1-defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H4MPT)-dependent formaldehyde oxidation . These results suggest that this gene, dmrA, may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H4MPT biosynthesis .
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