Biomedical subjects

Masaki Shintani

Publications and source records attributed to Masaki Shintani.

9 recordsLinked to original sources

PubMed2026

VicMAG, an open-source tool for visualizing circular metagenome-assembled genomes highlighting bacterial virulence and antimicrobial resistance.

Bacterial pathogens spread in clinical and environmental settings, and mobile genetic elements (MGEs), such as plasmids and phages, mediate the transfer of virulence factor genes (VFGs) and antimicrobial resistance genes (ARGs) among bacterial communities. Metagenomic analysis of environmental and wastewater samples using highly accurate long-read sequencing technologies, such as Pacific Biosciences (PacBio) HiFi sequencing, provides valuable insights into monitoring the regional spread of VFGs and ARGs, including dissemination mediated by MGEs. No visualization tool is currently available for the comprehensive display of numerous resulting circular metagenome-assembled genomes (cMAGs) with functional gene annotations. Here, we developed visualization of circular metagenome-assembled genome (VicMAG), a visualization tool for highly complex cMAGs derived from long-read metagenome assemblies annotated using updated databases of VFGs, ARGs, and MGEs. Using 353 cMAGs from PacBio HiFi sequencing of a wastewater sample, we demonstrated the utility of VicMAG for metagenome visualization. VicMAG provides comprehensive, size-aware visualization of cMAGs representing bacterial chromosomes and plasmids, annotated with VFGs, ARGs, and phages. By simultaneously visualizing all cMAGs in a framework, VicMAG facilitates a holistic understanding of the distribution and genomic context of VFGs and ARGs across complex microbial communities. This tool supports integrated surveillance of bacteria associated with virulence and antimicrobial resistance across clinical, environmental, and One Health contexts.

Metagenome↗

PubMed2006

The Sphingomonas plasmid pCAR3 is involved in complete mineralization of carbazole.

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.

Base Sequence↗

PubMed2006

Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation.

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 x 10(-1) and 3 x 10(-3) per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.

Bacterial Proteins↗

PubMed2005

Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains.

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.

Bacterial Proteins↗

PubMed2004

Large plasmid pCAR2 and class II transposon Tn4676 are functional mobile genetic elements to distribute the carbazole/dioxin-degradative car gene cluster in different bacteria.

The carbazole-catabolic plasmid pCAR1 isolated from Pseudomonas resinovorans strain CA10 was sequenced in its entirety; and it was found that pCAR1 carries the class II transposon Tn4676 containing carbazole-degradative genes. In this study, a new plasmid designated pCAR2 was isolated from P. putida strain HS01 that was a transconjugant from mating between the carbazole-degrader Pseudomonas sp. strain K23 and P. putida strain DS1. Southern hybridization and nucleotide sequence analysis of pCAR1 and pCAR2 revealed that the whole backbone structure was very similar in each. Plasmid pCAR2 was self-transmissible, because it was transferred from strain HS01 to P. fluorescens strain IAM12022 at the frequency of 2 x 10(-7) per recipient cell. After the serial transfer of strain HS01 on rich medium, we detected the transposition of Tn4676 from pCAR2 to the HS01 chromosome. The chromosome-located copy of Tn4676 was flanked by a 6-bp target duplication, 5'-AACATC-3'. These results experimentally demonstrated the transferability of pCAR2 and the functionality of Tn4676 on pCAR2. It was clearly shown that plasmid pCAR2 and transposon Tn4676 are active mobile genetic elements that can mediate the horizontal transfer of genes for the catabolism of carbazole.

Bacterial Proteins↗

PubMed2004

Divergent structures of carbazole degradative car operons isolated from gram-negative bacteria.

Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Amino Acid Sequence↗

PubMed2003

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676.

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.

Base Composition↗

PubMed2003

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676.

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed > 99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.

Amino Acid Sequence↗

PubMed2002

Organization and transcriptional characterization of catechol degradation genes involved in carbazole degradation by Pseudomonas resinovorans strain CA10.

Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon. Cat proteins of strain CA10 were very similar to those of P. putida, although the relatedness in non-coding regions was not high. It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.

Bacterial Proteins↗