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Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution. Kensuke Furukawa, 2004. Refining Our Perception of Bacterial Surfaces with the Atomic Force Microscope. Yves F. Dufrêne, 2004. Identification of a DtxR-Regulated Operon That Is Essential for Siderophore-Dependent Iron Uptake in Corynebacterium diphtheriae. Yilei Qian, 2002.The diphtheria toxin repressor (DtxR) uses Fe2+ as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae . A new IRP, designated IRP6, was cloned from C . diphtheriae by a SELEX-like procedure . DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent . The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria . IRP1 and IRP6 were repressed under high-iron conditions in wild-type C . diphtheriae C7(ß), but they were expressed constitutively in C7(ß) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake . A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains . Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively . The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product . Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele . These findings demonstrated that the irp6 operon in C . diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon . Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage. Eric S. Miller, 2003.The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined . The genome sequence is 244,835 bp, with an overall G+C content of 42.6% . It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators . Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp . While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4 . At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity) . The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40 . There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes . KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns . KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified . There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40 . From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages . Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved . CheZ-Mediated Dephosphorylation of the Escherichia coli Chemotaxis Response Regulator CheY: Role for CheY Glutamate 89. Ruth E. Silversmith, 2003.The swimming behavior of Escherichia coli at any moment is dictated by the intracellular concentration of the phosphorylated form of the chemotaxis response regulator CheY, which binds to the base of the flagellar motor . CheY is phosphorylated on Asp57 by the sensor kinase CheA and dephosphorylated by CheZ . Previous work (Silversmith et al., J . Biol . Chem . 276:18478, 2001) demonstrated that replacement of CheY Asn59 with arginine resulted in extreme resistance to dephosphorylation by CheZ despite proficient binding to CheZ . Here we present the X-ray crystal structure of CheYN59R in a complex with Mn2+ and the stable phosphoryl analogue BeF3- . The overall folding and active site architecture are nearly identical to those of the analogous complex containing wild-type CheY . The notable exception is the introduction of a salt bridge between Arg59 (on the ß3  3 loop) and Glu89 (on the ß44 loop) . Modeling this structure into the (CheY-BeF3--Mg2+)2CheZ2 structure demonstrated that the conformation of Arg59 should not obstruct entry of the CheZ catalytic residue Gln147 into the active site of CheY, eliminating steric interference as a mechanism for CheZ resistance . However, both CheYE89A and CheYE89Q, like CheYN59R, conferred clockwise flagellar rotation phenotypes in strains which lacked wild-type CheY and displayed considerable ( 40-fold) resistance to dephosphorylation by CheZ . CheYE89A and CheYE89Q had autophosphorylation and autodephosphorylation properties similar to those of wild-type CheY and could bind to CheZ with wild-type affinity . Therefore, removal of Glu89 resulted specifically in CheZ resistance, suggesting that CheY Glu89 plays a role in CheZ-mediated dephosphorylation . The CheZ resistance of CheYN59R can thus be largely explained by the formation of the salt bridge between Arg59 and Glu89, which prevents Glu89 from executing its role in catalysis .
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