Koshiro Miura

Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.

Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.

The rpoS gene regulates OP2, an operon for the lower pathway of xylene catabolism on the TOL plasmid, and the stress response in Pseudomonas putida mt-2

Abstract Operon OP2 on the Pseudomonas putida TOL plasmid encodes enzymes for m-toluate catabolism; transcription of this operon is activated by XylS in the presence of m-toluate. Because transcriptional activation of OP2 specifically occurs in the stationary phase of growth both in P. putida and in Escherichia coli, we suspected that its transcription is dependent on RpoS (σS). Therefore, we constructed a rpoS disruption strain of P. putida mt-2, and assayed OP2 expression and other phenotypes. OP2 transcription was dependent on rpoS, indicating that the stationary-phase specific activation of OP2 is due to positive regulation by RpoS in P. putida mt-2. The rpoS mutant exhibited reduced viability during the stationary phase and was sensitive to high salt concentrations and H2O2. P. putida mt-2 has two catalase isozymes, KatA and KatB. Expression of the katB gene was specific to the stationary phase and entirely dependent on the rpoS gene, while the katA gene expressed during log phase partially required rpoS. There were no significant changes in tolerance to high temperature or UV light in the rpoS mutant. No difference was observed between the E. colirpoS mutants and their parents in their capacity to activate OP2, suggesting that the mechanism of OP2 activation in the stationary phase of growth differs between P. putida and E. coli.

Protein binding in vivo to OP2 promoter of the Pseudomonas putida TOL plasmid.

The transcription of OP2 encoding enzymes for m‐toluate catabolism on the Pseudomonasputida TOL plasmid is activated by basal‐level XylS protein in the presence of m‐toluate or by overproduced XylS protein in the absence of m‐toluate. In this study, in vivo dimethyl sulfate (DMS) footprinting was performed to understand the mechanism of transcriptional regulation of OP2 promoter by XylS. In the presence of overproduced XylS without m‐toluate, several protected nucleotides were observed, indicating the binding of RNA polymerase to DNA. However, the protection was canceled upon addition of m‐toluate. These results suggest that RNA polymerase is retained by XylS on the OP2 promoter in the absence of inducer, and is released by m‐toluate binding to XylS, concomitant with transcription.

Genome-wide Analysis of Chlamydophila pneumoniae Gene Expression at the Late Stage of Infection

Chlamydophila pneumoniae, an obligate intracellular eubacterium, changes its form from a vegetative reticulate body into an infectious elementary body during the late stage of its infection cycle. Comprehension of the molecular events in the morphological change is important to understand the switching mechanism between acute and chronic infection, which is deemed to relate to the pathogenesis of atherosclerosis. Herein, we have attempted to screen genes expressed in the late stage with a genome-wide DNA microarray, resulting in nomination of 17 genes as the late-stage genes. Fourteen of the 17 genes and six other genes predicted as late-stage genes were confirmed to be up-regulated in the late stage with a quantitative reverse transcriptase–polymerase chain reaction. These 20 late-stage genes were classified into two groups by clustering analysis: ‘drastically induced’ and ‘moderately induced’ genes. Out of eight drastically induced genes, four contain σ28 promoter-like sequences and the other four contain an upstream common sequence. It suggests that besides σ28, there are certain up-regulatory mechanisms at the late stage, which may be involved in the chlamydial morphological change and thus pathogenesis.

sup 14 C( p , n ) sup 14 N reaction at E sub p =35 MeV

Differential cross sections for the {sup 14}C({ital p},{ital n}){sup 14}N reaction were measured at {ital E}{sub {ital p}}=35 MeV. A number of spin-isospin excitations have been observed including Gamow-Teller-type 0{sup +}{r arrow}1{sup +} and 0{h bar}{omega} and 1{h bar}{omega} jump stretched transitions. Distorted-wave Born-approximation calculations using shell-model wave functions have successfully reproduced the experimental results. Renormalization factors of about 0.5 were required for the spin-flip transitions leading to the 3.947-MeV 1{sup +} state and to the 7.026-MeV 2{sup +} state, where {Delta}{ital J}({Delta}{ital L},{Delta}{ital S})=1(0,1) and 2(2,1), respectively. Proton and neutron optical-potential parameters were derived in the course of the present work.

Imaging of IHF‐induced DNA Bending Structure by AFM

Integration host factor (IHF) of Escherichia coli is a DNA-bending protein. Although first discovered as a host factor for bacteriophage λ integration, IHF functions in many processes that involve higher order protein-DNA complexes: e.g., in replication, where it binds to ori C; in transcriptional regulation, where it binds upstream of many σ-dependent promoters; and in a variety of site-specific recombination systems. The primary function of IHF appears to be architectural, i.e., introducing a sharp bend in the DNA that facilitates the interaction of other components in a nucleoprotein array. In this study, the structure of the DNA-IHF nucleoprotein complex in the OP1 promoter region on the Pseudomonas TOL plasmid was analyzed using atomic force microscopy (AFM), which can directly visualize biological structure under near-native conditions without crystallization. We found that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription. The DNA fragment containing the OP1 promoter was purified from the palsmid, and it was amplified by PCR. The IHF protein was purified from E. coli strain D1210HP transformed with the plasmid pPL-hip him A-5. The IHF-DNA complexes were formed by mixing the both of solutions, and proteins not reacted with DNA fragments were removed by filtration. The NanoScopeIIIa Multi Mode system operated in tapping mode was used for observation of IHF-DNA complexes. The AFM image of the IHF-DNA complexes showed a sharp DNA bend at the IHF-binding site on the fragment. This sharp DNA bend can be used as a clear landmark of IHF-binding site on the templates. To investigate whether the binding of IHF to the IHF binding site induced bending of the template, a statistical analysis on images of IHF-DNA complexes was performed. Measurements of the DNA bending angles yielded a distribution with the mean bend angle of 123 °. Contrary, uncomplexed DNA molecules showed Gaussian distribution centered at 0 °, indicating no intrinsic curvature of the DNA fragments at these locations. This suggests that the bending observed on the complexed DNA molecules was dependent on the binding of IHF. The observed bend-angle distribution in the absence of IHF is to be accounted for by random thermal fluctuations. As shown in this study, AFM will be a useful tool for visualizing the effects of protein-DNA interactions and contribute to a better understanding of the complex mechanisms of transcriptional regulation.