Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

SummaryPlasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazoledegradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistancenodulation-cell division family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared with pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.

Nuclear factor-kappaB (NF-kappaB) plays a key role in regulating expression of several genes involved in the pathophysiology of endotoxic shock. We investigated whether in vivo introduction of synthetic double-stranded DNA with high affinity for the NF-kappaB binding site could block expression of genes mediating pulmonary vascular permeation and thereby provide effective therapy for septic lung failure. Endotoxic shock was induced by an intravenous injection of 10 mg/kg Escherichia coli endotoxin in mice. We introduced NF-kappaB decoy oligodeoxynucleotide (ODN) in vivo 1 h after endotoxic shock by using a gene transfer kit. At 10 h, blood samples were collected for measurement of histamine and for blood-gas analysis. Gene and protein expression levels of target molecules were determined by means of Northern and Western blot analyses, respectively. The transpulmonary flux of (125)I-labeled albumin was used as an index of lung vascular permeability. Administration of endotoxin caused marked increases in plasma histamine and gene and protein expressions of histidine decarboxylase, histamine H(1) receptors, and inducible nitric oxide synthase in lung tissues. Elevated lung vascular permeability was also found. Blood-gas analysis showed concurrent decreases in arterial Po(2), Pco(2), and pH. All of these events induced by endotoxin were significantly inhibited by transfection of NF-kappaB decoy ODN but not by its mutated (scrambled) form (used as a control). Our results indicate for the first time the potential usefulness of NF-kappaB decoy ODN for gene therapy of endotoxic shock.

The performance of Fourier-transform profilometry is enhanced by a new technique that is based on spatial frequency multiplexing combined with the Gushov-Solodkin phase unwrapping algorithm. The technique permits the three-dimensional shape measurement of objects that have discontinuous height steps and/or spatially isolated surfaces, which has not been possible by conventional Fourier-transform profilometry. An important feature of the technique is that it requires only a single fringe pattern; the single-shot recording makes possible the instantaneous three-dimensional shape measurement of discontinuous objects in fast motion. Experimental results are presented that demonstrate the validity of the principle.

The impacts of plasmid carriage on the host cell were comprehensively analysed using the conjugative plasmid pCAR1 in three different Pseudomonas hosts, P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Plasmid carriage reduced host fitness, swimming motility, and resistance to osmotic or pH stress. Plasmid carriage brought about alterations in primary metabolic capacities in the TCA cycle of the hosts. Differentially transcribed genes in the three hosts associated with plasmid carriage were identified by growth phase-dependent transcriptome analyses. Plasmid carriage commonly showed a greater effect on the host transcriptome at the transition and early stationary phases. The transcriptome alterations were similar between KT2440 and PAO1. Transcriptions of numbers of genes encoding ribosomal proteins, F-type ATPase, and RNAP core in both strains were not suppressed enough in the early stationary phase by plasmid carriage. These responses may have been responsible for the reduction in host fitness, motility and stress resistances. Host-specific responses to plasmid carriage were transcriptional changes of genes on putative prophage or foreign DNA regions. The extents of the impacts on host phenotypes and transcriptomes were similarly greatest in KT2440 and lowest in Pf0-1. These findings suggest that host cell function was actively regulated by plasmid carriage.

We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazoledegradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole-and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazoledegradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.Horizontal gene transfer plays an important role in the evolution and adaptation of prokaryotes (12,40). Plasmids are extrachromosomal elements that are replicated autonomously and provide beneficial functions to their hosts, such as resistance to antibiotics and degradation of recalcitrant aromatic compounds. The essential functions encoded on plasmid backbones, e.g., replication initiation, active partitioning, and conjugative transfer, are partially dependent on host factors (1). For example, De Gelder et al. (5) showed that the host range of the multiresistance plasmid pB10 was influenced by donor strains within an activated-sludge microbial community. Accessory genes are also regulated by host factors, and there are reports showing that catabolic operons on plasmids require host regulatory elements (21, 28). Although these results suggest the importance of the host in imparting plasmid function, additional investigations are necessary to understand the mechanisms of plasmid survival and to determine how plasmid dynamics are affected by hosts with different types of genomic organization.The carbazole-catabolic plasmid pCAR1 was discovered in Pseudomonas resinovorans CA10 (27) and was the first IncP-7 plasmid to be completely sequenced (20,26,36). Carbazole is a structural analog of dioxin...

Thermophile Hydrogenobacter thermophilus cytochrome c(552) (HT) is a stable protein with denaturation temperatures (T(m)) of 109.8 and 129.7 degrees C for the oxidized and reduced forms, respectively [Uchiyama, S., Ohshima, A., Nakamura, S., Hasegawa, J., Terui, N., Takayama, S. J., Yamamoto, Y., Sambongi, Y., and Kobayashi, Y. (2004) J. Am. Chem. Soc. 126, 14684-14685]. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the T(m) value of the oxidized form by approximately 6 degrees C, the T(m) value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of approximately 20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering.

Histamine is highly concentrated in the heart of animals and humans. Excessive release in pathophysiological conditions, such as immediate hypersensitivity and septic shock, causes cardiac dysfunction and arrhythmias. Previous pharmacological studies revealed that H 1 and H 2 receptors mediate these effects. Yet, an accurate estimate of the distribution and molecular characteristics of cardiac histamine receptors is missing. Recently, the genes encoding H 1 and H 2 receptors have been cloned, and the amino acid sequence and protein structure have been elucidated. Accordingly, we analyzed gene and protein expression levels of H 1 and H 2 receptors in atria and ventricles of guinea pig, rabbit, rat, and human hearts. With immunocytochemical techniques, we examined the regional expression of H 1 and H 2 receptor proteins in the sinoatrial and atrioventricular nodes and surrounding myocardium of the guinea pig heart. Northern and Western blot studies revealed that cardiac histamine H 1 and H 2 receptors are variably distributed among different mammalian species and different regions of the heart, whereas H 2 receptors are abundantly expressed in human atrial and ventricular myocardium. These findings agree with those of previous pharmacological studies, clearly demonstrating that the responses of the heart to histamine depend on the expression level of H 1 and H 2 receptors. The highly abundant expression of H 2 receptors in the human heart substantiates histamine arrhythmogenicity in various disease states. The new knowledge of a differential distribution of histamine receptor subtypes in the human heart will foster a better understanding of histamine roles in cardiovascular pathophysiology and may contribute to new therapeutic approaches to histamine-induced cardiac dysfunctions.The actions of histamine as a cardiac stimulant have been appreciated for more than 90 years since the seminal work of Dale and Laidlaw (1910). The primary and direct actions of histamine in the heart are characterized by an increase in sinus rate and ventricular automaticity, a decrease in AV conduction velocity, and an increase in force of contraction (Levi et al., 1991;Hattori, 1999). Because histamine is present in high concentrations in cardiac tissues in most animal species, including humans (Bristow et al., 1982;Wolff and Levi, 1986), its release from cardiac stores and its subsequent actions on the heart may be of importance in pathological conditions associated with histamine release (Levi, 1988). In this context, we recently reported that superinduction of histamine receptor gene products and histamine overproduction contribute to the hemodynamic derangement characteristic of septic shock (Matsuda et al., 2002).The negative dromotropic and positive chronotropic effects of histamine invariably result from the activation of H 1 and H 2 receptors, respectively (Levi et al., 1991;Hattori, 1999). In contrast, there is a marked species difference in the subtype of histamine receptors mediating the positive inotropic effect. Mor...