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Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the Caulobacter FliF Motor Protein. Björn Grünenfelder, 2004.Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus . The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release . Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly . To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF . Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein . Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously . However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability . This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence . The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF . Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms . Degradation of Polycyclic Aromatic Hydrocarbons by a Newly Discovered Enteric Bacterium, Leclercia adecarboxylata. Priyangshu Manab Sarma, 2004.A bacterial strain, PS4040, capable of degrading polycyclic aromatic hydrocarbons for use as the sole carbon source was isolated from oily-sludge-contaminated soil . The 16S rRNA gene showed 98.8% homology to that of Leclercia adecarboxylata . Comparative molecular typing with the clinical strain of L . adecarboxylata revealed that there were few comigrating and few distinct amplimers among them . Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp . Strain HW13 Isolated from a Pristine Environment. Wataru Kitagawa, 2002.The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria . A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp . strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals . The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence . The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively . They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R . eutropha JMP134 . The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134 . Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives . Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not . Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC . Perfringolysin O Expression in Clostridium perfringens Is Independent of the Upstream pfoR Gene. Milena M. Awad, 2002.The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O . Previous studies (T . Shimizu, A . Okabe, J . Minami, and H . Hayashi, Infect . Immun . 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene . In an attempt to confirm this hypothesis in C . perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA+ shuttle plasmids that varied only in their ability to encode an intact pfoR gene . No difference in the ability to produce perfringolysin O was observed for C . perfringens strains carrying these plasmids . In addition, chromosomal pfoR mutants were constructed by homologous recombination in C . perfringens . Again no difference in perfringolysin O activity was observed . Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C . perfringens .
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