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ExbB and ExbD Do Not Function Independently in TonB-Dependent Energy Transduction. Kiara G. Held, 2002.ExbB and ExbD proteins are part of the TonB-dependent energy transduction system and are encoded by the exb operon in Escherichia coli . TonB, the energy transducer, appears to go through a cycle during energy transduction, with the absence of both ExbB and ExbD creating blocks at two points: (i) in the inability of TonB to respond to the cytoplasmic membrane proton motive force and (ii) in the conversion of TonB from a high-affinity outer membrane association to a high-affinity cytoplasmic membrane association . The recent observation that ExbB exists in 3.5-fold molar excess relative to the molarity of ExbD in E . coli suggests the possibility of two types of complexes, those containing both ExbB and ExbD and those containing only ExbB . Such distinct complexes might individually manifest one of the two activities described above . In the present study this hypothesis was tested and rejected . Specifically, both ExbB and ExbD were found to be required for TonB to conformationally respond to proton motive force . Both ExbB and ExbD were also required for association of TonB with the cytoplasmic membrane . Together, these results support an alternative model where all of the ExbB in the cell occurs in complex with all of the ExbD in the cell . Based on recently determined cellular ratios of TonB system proteins, these results suggest the existence of a cytoplasmic membrane complex that may be as large as 520 kDa . AmfS, an Extracellular Peptidic Morphogen in Streptomyces griseus. Kenji Ueda, 2002.The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus . The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames . A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule . Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain . A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS . A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen . The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the ß' Subunit of RNA Polymerase. Susanne Mahren, 2003.Transcription of the ferric citrate transport system of Escherichia coli K-12 is mediated by the extracytoplasmic-function (ECF) sigma factor FecI, which is activated by ferric citrate in the growth medium . By using a bacterial two-hybrid system, it was shown in vivo that FecI binds to the ß' subunit of RNA polymerase . The inactive mutant protein FecI(K155E) displayed reduced binding to ß', and small deletions along the entire FecI protein led to total impairment of ß' binding . In vitro, FecI was retained on Ni2+-nitrilotriacetic acid agarose loaded with a His-tagged ß'1-313 fragment and coeluted with ß'1-313 . Binding of FecI to ß' and ß'1-313 was enhanced by FecR1-85, which represents the cytoplasmic portion of the FecR protein that transmits the inducing signal across the cytoplasmic membrane . Interaction of FecR with FecI was demonstrated by showing that isolated FecR inhibited degradation of FecI by trypsin . This is the first demonstration of binding of an ECF sigma factor of the FecI type to the ß' subunit of RNA polymerase and of binding being enhanced by the protein that activates the ECF sigma factor . Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences. Jorge L. M. Rodrigues, 2003.The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates . Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X . fastidiosa isolated from different plant hosts . A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains . Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity . Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity . A multiplex PCR protocol was developed and its sensitivity tested . Sequencing of PCR products confirmed the validity of the multiplex PCR . Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants . Specific detection of X . fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease .
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