Kazuhiro Matsui

Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

ABSTRACT The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a “transient” host of pCAR1.

Behavior of the IncP-7 carbazole-degradative plasmid pCAR1 in artificial environmental samples

In artificial environmental samples, the behavior of the IncP-7 conjugative plasmid pCAR1, which is involved in the catabolism of carbazole, was monitored. Sterile soil and water samples supplemented with carbazole were prepared. After inoculation with Pseudomonas putida harboring pCAR1, seven species of the genus Pseudomonas, and three other bacterial species, were monitored for carbazole degradation, bacterial survival, and conjugative transfer of pCAR1. In artificial soils, more than 90% of the carbazole was degraded in samples with high water content, suggesting that the water content is a key factor in carbazole degradation in artificial soils. In three of the artificial environmental water samples, more than 95% of the carbazole was degraded. Transconjugants were detected in some artificial water samples, but not in the artificial soil samples, suggesting that pCAR1 is preferably transferred in aqueous environments. Composition analysis of the artificial water samples and examination of conjugative transfer indicated that the presence of the divalent cations Ca2+ and Mg2+ promoted the plasmid transfer. The presence of carbazole also increases in incidence of transconjugants, probably by enhancing their growth. In contrast, humic acids in the liquid layer of artificial soil samples appeared to prevent conjugative transfer.

Effects of three different nucleoid-associated proteins encoded on IncP-7 plasmid pCAR1 on host Pseudomonas putida KT2440.

ABSTRACT Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities.

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.

A new method for absolute accuracy evaluation of a US-guided HIFU system with heterogeneous phantom

High Intensity Focused Ultrasound as a non-invasive treatment method has gaining its popularity in various clinical application. HIFU is usually used with navigation system with which to make pre-operative plan. Our group developed a US-guided HIFU system for breast cancer treatment. In order to precisely remove the tumor region and reduce the damage to the surrounding normal tissue to minimal level, it is important to evaluate the absolute accuracy of the treatment system, i.e. the spatial difference between the planned target region and the actual treated one. However, this task is often hard and there is few related work. In this research, we provide a method to quantitatively evaluate the accuracy of the US-guided HIFU treatment on 2D treatment plane. We use heterogeneous phantom which contains chicken meat surrounded by porcine liver to mimic tumor surrounded by normal tissue, and 3D printed markers to serve as spatial reference. The simulated tumor and simulated surrounding normal tissue as well as the markers are embedded into transparent gel with breast shape. With this setting, the boundary and shape of the treated region can be preserved. Moreover, the planned treatment region on the US image and the actual treated region can be registered into the same reference coordinate frame for comparison. The result demonstrates that the proposed method is capable of evaluating the absolute accuracy of the US-guided HIFU treatment on 2D treatment plane and our US-guided HIFU system has achieved high accuracy.

Preclinical ultrasound image-guided high intensity focused ultrasound robot system for breast cancer therapy.

Ultrasound imaging provides real-time feedback for highly accurate positioning and dosing control. In addition, spatial restriction of high-intensity focused ultrasound (HIFU) transducer position in the ultrasound image-guided system is less than that in the case of MRI-guided system. Therefore, wider beam approaching path can be used in the ultrasound image-guided system. To shorten the total treatment time, reducing the cooling time between sonication intervals is essential. Using a wide approach path promotes a reduction in both the cooling time and the risk of heat deposition to the body surface. The array in our preclinical HIFU system is supported by a 5-axis robotic system that enables motion with a pivot fixed at the focal point. In this report, we describe a HIFU beam imaging system that provides highly accurate pivoting motion and coagulation monitoring in real-time for dose control during HIFU treatment.

Divalent cations increase the conjugation efficiency of the incompatibility P-7 group plasmid pCAR1 among different Pseudomonas hosts

The incompatibility (Inc) P-7 group plasmid pCAR1 can be efficiently transferred among bacteria in artificial microcosms in the presence of divalent cations Ca2+ and Mg2+. One-on-one mating assays between Pseudomonas strains with different plasmids showed that the promotion of conjugation efficiency by divalent cations was exhibited in other plasmids, including pB10 and NAH7; however, this effect was larger in IncP-7 plasmids. The impact on pCAR1 conjugation differed according to donor-recipient pairs, and conjugation efficiency promotion was clearly detected between the donors P. resinovorans CA10dm4 and P. fluorescens Pf0-1 and the recipients P. putida KT2440 and CA10dm4. Transcriptome analyses showed that pCAR1 gene expression did not respond to cation changes, including the tra/trh genes involved in its transfer. However, the transcription of oprH genes, encoding putative outer-membrane proteins in both the donor and the recipient, were commonly upregulated under cation-limited conditions. The conjugation frequency of pCAR1 in the KT2440 oprH mutant was found not to respond to cations. This effect was partially recovered by complementation with the oprH gene, suggesting that OprH is involved in the increase of pCAR1 conjugation efficiency by divalent cations.

Self-shape estimation algorithm for a flexible ultrasonic array probe

A self-shape estimation algorithm for a flexible ultrasonic array probe was described in the paper. Position information of each element in the array is essentially required to achieve a focal control in ultrasonic imaging process. The purpose of this study is to develop an algorithm to estimate array shape without other sensors. In our proposed algorithm, beam image (BI) was used as an evaluation function in the estimation of the shape. BI is an image representing a transmitted beam profile in the imaging object. BI was obtained by scanning of receive focal points around the transmit focal point. The quality factor of BI was used as an evaluation function and parameters describing assumed shapes were searched. We conducted simulation and gel experiments with commercially available flexible probe unit. The algorithm was evaluated by the quality of an estimated self-shape and reconstructed images using an estimated self-shape. The theoretical lateral and depth resolutions were 0.5~1mm and 0.3mm in light of...

Visualization of 3D temperature distribution caused by exposure of HIFU with thermo-chromic liquid crystal phantom

HIFU (High Intensity Focused Ultrasound) is one of a promising minimally invasive treatment method. It is expected that energy concentration causes heat coagulation around focal area, while dispersion does not give a damage outside focus area. To establish temperature control technique, it is important to investigate three dimensional temperature around the target. For this purpose we developed a temperature distribution measurement visualization method. In clinical practice, visualization of temperature distribution produced by clinical therapeutic ultrasonic devices is useful for ensuring clinical efficacy and patient safety. In this study, 3D temperature measurement phantom with MTLC (micro-capsuled thermo-chromic liquid crystal) was proposed, and its performance was demonstrated.