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In response to the International Commission on Radiological Protection, which lowered the lens equivalent dose limit, Japan lowered the lens dose limit from 150 mSv y -1 to 100 mSv/5 years and 50 mSv y -1 , with this new rule taking effect on 1 April 2021. DOSIRIS ® is a dosimeter that can accurately measure lens dose. Herein, we investigated lens dose in interventional cardiology physicians 1 year before and after the reduction of the lens dose limit using a neck dosimeter and lens dosimeter measurements. With an increase in the number of cases, both personal dose equivalent at 0.07 mm depth [Hp(0.07), neck dosimeter] and personal dose equivalent at 3 mm depth [Hp(3), lens dosimeter] increased for most of the physicians. The Hp(3) of the lens considering the shielding effect of the Pb glasses using lens dosimeter exceeded 20 mSv y -1 for two of the 14 physicians. Protection from radiation dose will become even more important in the future, as these two physicians may experience radiation dose exceeding 100 mSv/5 years. The average dose per procedure increased, but not significantly. There was a strong correlation between the neck dosimeter and lens dosimeter scores, although there was no significant change before and after the lens dose limit was lowered. This correlation was particularly strong for physicians who primarily treated patients. As such, it is possible to infer accurate lens doses from neck doses in physicians who primarily perform diagnostics. However, it is desirable to use a dosimeter that can directly measure Hp(3) because of the high lens dose., (© 2024 Society for Radiological Protection. Published on behalf of SRP by IOP Publishing Limited. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Although interventional radiology (IVR) is preferred over surgical procedures because it is less invasive, it results in increased radiation exposure due to long fluoroscopy times and the need for frequent imaging. Nurses engaged in cardiac IVR receive the highest lens radiation doses among medical workers, after physicians. Hence, it is important to measure the lens exposure of IVR nurses accurately. Very few studies have evaluated IVR nurse lens doses using direct dosimeters. This study was conducted using direct eye dosimeters to determine the occupational eye dose of nurses engaged in cardiac IVR, and to identify simple and accurate methods to evaluate the lens dose received by nurses. Over 6 months, in a catheterization laboratory, we measured the occupational dose to the eyes (3 mm dose equivalent) and neck (0.07 mm dose equivalent) of nurses on the right and left sides. We investigated the relationship between lens and neck doses, and found a significant correlation. Hence, it may be possible to estimate the lens dose from the neck badge dose. We also evaluated the appropriate position (left or right) of eye dosimeters for IVR nurses. Although there was little difference between the mean doses to the right and left eyes, that to the right eye was slightly higher. In addition, we investigated whether it is possible to estimate doses received by IVR nurses from patient dose parameters. There were significant correlations between the measured doses to the neck and lens, and the patient dose parameters (fluoroscopy time and air kerma), implying that these parameters could be used to estimate the lens dose. However, it may be difficult to determine the lens dose of IVR nurses accurately from neck badges or patient dose parameters because of variation in the behaviors of nurses and the procedure type. Therefore, neck doses and patient dose parameters do not correlate well with the radiation eye doses of individual IVR nurses measured by personal eye dosimeters. For IVR nurses with higher eye doses, more accurate measurement of the radiation doses is required. We recommend that a lens dosimeter be worn near the eyes to measure the lens dose to IVR nurses accurately, especially those exposed to relatively high doses.

Mobile radiography allows for the diagnostic imaging of patients who cannot move to the X-ray examination room. Therefore, mobile X-ray equipment is useful for patients who have difficulty with movement. However, staff are exposed to scattered radiation from the patient, and they can receive potentially harmful radiation doses during radiography. We estimated occupational exposure during mobile radiography using phantom measurements. Scattered radiation distribution during mobile radiography was investigated using a radiation survey meter. The efficacy of radiation-reducing methods for mobile radiography was also evaluated. The dose decreased as the distance from the X-ray center increased. When the distance was more than 150 cm, the dose decreased to less than 1 μSv. It is extremely important for radiological technologists (RTs) to maintain a sufficient distance from the patient to reduce radiation exposure. The spatial dose at eye-lens height increases when the bed height is high, and when the RT is short in stature and abdominal imaging is performed. Maintaining sufficient distance from the patient is also particularly effective in limiting radiation exposure of the eye lens. Our results suggest that the doses of radiation received by staff during mobile radiography are not significant when appropriate radiation protection is used. To reduce exposure, it is important to maintain a sufficient distance from the patient. Therefore, RTs should bear this is mind during mobile radiography.

The new recommendation of the International Commission on Radiological Protection for occupational eye dose is an equivalent dose limit to the eye of 20 mSv year-1, averaged over a 5-year period. This recommendation is a drastic reduction from the previous limit of 150 mSv year-1. Hence, it is important to protect physicians' eyes from X-ray radiation. Particularly in interventional radiology (IVR) procedures, many physicians use protective lead (Pb) glasses to reduce their occupational exposure. This study assessed the shielding effects of novel 0.07 mm Pb glasses. The novel glasses (XR-700) have Pb-acrylic lens molded in three dimensions. We studied the novel type of 0.07 mm Pb glasses over a period of seven consecutive months. The eye dose occupational radiation exposure of seven IVR physicians was evaluated during various procedures. All IVR physicians wore eye dosimeters (DOSIRIS™) close to the left side of the left eye. To calculate the shielding effects of the glasses, this same type of eye dosimeter was worn both inside and outside of the Pb lenses. The average shielding effect of the novel glasses across the seven physicians was 61.4%. Our results suggest an improved shielding effect for IVR physicians that use these glasses. No physician complained that the new glasses were uncomfortable; therefore comfort is not a problem. The lightweight glasses were acceptable to IVR physicians, who often must perform long procedures. Thus, the novel glasses are comfortable and reasonably protective. Based on the results of this study, we recommend that IVR physicians use these novel 0.07 mm Pb glasses to reduce their exposure., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

In recent years, endovascular treatment of aortic aneurysms has attracted considerable attention as a promising alternative to traditional surgery. Hybrid operating room systems (HORSs) are increasingly being used to perform endovascular procedures. The clinical benefits of endovascular treatments using HORSs are very clear, and these procedures are increasing in number. In procedures such as thoracic endovascular aortic repair (TEVAR) and endovascular aortic repair (EVAR), wires and catheters are used to deliver and deploy the stent graft in the thoracic/abdominal aorta under fluoroscopic control, including DSA. Thus, the radiation dose to the patient is an important issue. We determined radiation dose indicators (the dose-area product (DAP) and air karma (AK) parameters) associated with endovascular treatments (EVAR and TEVAR) using a HORS. As a result, the mean ± standard deviation (SD) DAPs of TEVAR and EVAR were 323.7 ± 161.0 and 371.3 ± 186.0 Gy x cm 2 , respectively. The mean ± SD AKs of TEVAR and EVAR were 0.92 ± 0.44 and 1.11 ± 0.54 Gy, respectively. The mean ± SD fluoroscopy times of TEVAR and EVAR were 13.4 ± 7.1 and 23.2 ± 11.7 min, respectively. Patient radiation dose results in this study of endovascular treatments using HORSs showed no deterministic radiation effects, such as skin injuries. However, radiation exposure during TEVAR and EVAR cannot be ignored. The radiation dose should be evaluated in HORSs during endovascular treatments. Reducing/optimizing the radiation dose to the patient in HORSs is important.

Although the clinical value of fluoroscopically guided respiratory endoscopy (bronchoscopy) is clear, there have been very few studies on the radiation dose received by staff during fluoroscopically guided bronchoscopy. The International Commission on Radiological Protection (ICRP) is suggesting reducing the occupational lens dose limit markedly from 150 to 20 mSv/year, averaged over defined periods of five years. The purpose of this study was to clarify the current occupational eye dose of bronchoscopy staff conducting fluoroscopically guided procedures. We measured the occupational eye doses (3-mm-dose equivalent, Hp(3)) of bronchoscopy staff (physicians and nurses) over a 6-month period. The eye doses of eight physicians and three nurses were recorded using a direct eye dosimeter, the DOSIRIS. We also estimated eye doses using personal dosimeters worn at the neck. The mean ± SD radiation eye doses (DOSIRIS) to physicians and nurses were 7.68 ± 5.27 and 2.41 ± 1.94 mSv/6 months, respectively. The new lens dose limit, 20 mSv/year, may be exceeded among bronchoscopy staff, especially physicians. The eye dose of bronchoscopy staff (both physicians and nurses) was underestimated when measured using a neck dosimeter. Hence, the occupational eye dose of bronchoscopy staff should be monitored. To reduce the occupational eye dose, we recommend that staff performing fluoroscopically guided bronchoscopy wear Pb glasses. correct evaluation of the lens dose [Hp(3)] using an eye dosimeter such as the DOSIRIS is necessary for bronchoscopy staff., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Monitoring and protecting of occupational eye doses in interventional radiology (IR) are very important matters. DOSIRIS™ is the useful solution to estimate the 3 mm dose-equivalent (Hp(3)), and it can be worn behind lead glasses. And DOSIRIS™, adjustable according to 3 axes, it is ideally placed as close to the eye and in contact with the skin. So, DOSIRIS™ will be suitable eye lens dosimeter. However, the fundamental characteristics of the DOSIRIS™ in the diagnostic x-ray energy domain (including that of IR x-ray systems) remain unclear. Here, we evaluated the performance of the dosimeter in that energy range. As a result, the DOSIRIS™ has good fundamental characteristics (batch uniformity, dose linearity, energy dependence, and angular dependence) in the diagnostic x-ray energy domain. We conclude that the DOSIRIS™ has satisfactory basic performance for occupational eye dosimetry in diagnostic x-ray energy settings (including IR x-ray systems).

A 6625-base pair transposon, Tn4556, was initially isolated from a Streptomyces strain and a sequence analysis was performed; however, its annotation data remain incomplete. At least three positions were identified as frameshift and base-exchange errors by resequencing. The revised sequence revealed that Tn4556 contains four open reading frames that encode transposase, methyltransferase, isoprenyl diphosphate transferase, and resolvase, respectively. Thirty-eight-base pair inverted repeat (IR) sequences at both ends contained a 1-bp mismatch flanked by a target duplication site, and transposition efficiency was improved by the replacement of imperfectly matched IR-L to perfectly matched IR-L. The detection of Tn4556 transposition was markedly facilitated using a delivery vector carrying a strictly counter-selectable marker for Streptomyces strains.

Background: Genetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces., Results: To determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of D-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by D-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions., Conclusion: In this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.

Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.

It is important to measure the radiation dose [3-mm dose equivalent, Hp(3)] in the eye. This study was to determine the current occupational radiation eye dose of staff conducting interventional cardiology procedures, using a novel direct eye dosimeter. We measured the occupational eye dose [Hp(3)] in physicians and nurses in a catheterization laboratory for 6-months. The eye doses [Hp(3)] of 12 physicians (9 with Pb glasses, 3 without), and 11 nurses were recorded using a novel direct eye dosimeter, the DOSIRIS TM . We placed dosimeters above and under the glasses. We also estimated the eye dose [0.07-mm dose equivalent] using a neck personal dosimeter. The eye doses among interventional staff ranked in the following order: physicians without Pb glasses > physicians with Pb glasses > nurses. The shielding effect of the glasses (0.07-mm Pb) in a clinical setting was approximately 60%. In physicians who do not wear Pb glasses, the eye dose may exceed the new regulatory limit for IR staff. We found good correlations between the neck dosimeter dose and eye dosimeter dose (inside or outside glasses, R 2  = 0.93 and R 2  = 0.86, respectively) in physicians. We recommend that interventional physicians use an eye dosimeter for correct evaluation of the lens dose.

Objective: To evaluate an analog library of betaine-type cellular metabolites, which are naturally found in polar fish for survival in subzero temperatures, for preventing denaturation of enzymes during freezing., Results: Comparison of the cryoprotective ability of reported cryoprotectants, such as dimethylsulfoxide, glycerol, ectoine, hydroxyectoine, and trehalose, with betaine-type analogs using α-glucosidase revealed that analogs introducing C3-C6 alkyl chains into an ammonium cation retained 20 % higher activity than the control cryoprotectants at the same concentration. In particular, the analog possessing triplicate n-butyl chains showed a profound effect. It allowed retention of enzyme activity to 95 % even after 100 freeze-thaw cycles, while addition of the control cryoprotectants decreased the activity to 10-20 %. The cryoprotective ability of betaine-type analogs can be applied not only to α-glucosidase but also other enzymes such as β-glucosidase, alkaline phosphatase, lactose dehydrogenase, sulfatase, and horseradish peroxidase., Conclusion: Synthetic betaine-type metabolite analogs possess practicable cryoprotective ability for various enzymes, and are considerably superior to previously reported cryoprotectants.

The end products from starch hydrolysis by amylases have important applications in various industries. Here, two amylases derived from two Streptomyces species that hydrolyze soluble starch from potato produced maltotriose as the primary maltooligosaccharide product. The genes, annotated as putative glycoside hydrolases, were cloned and expressed in Streptomyces lividans. These amylases displayed hydrolysis activity from pH 3 to 9.5 and were not affected by Ca(2+.) Optimal production of maltotriose was between 20 and 30 °C at pH 6.5. At the optimal temperature, both amylases produced maltotriose-rich end products rather than either maltose or maltotetraose.

The ability of bacterial plasmids to adapt to novel hosts and thereby shift their host range is key to their long-term persistence in bacterial communities. Promiscuous plasmids of the incompatibility group P (IncP)-1 can colonize a wide range of hosts, but it is not known if and how they can contract, shift or further expand their host range. To understand the evolutionary mechanisms of host range shifts of IncP-1 plasmids, an IncP-1β mini-replicon was experimentally evolved in four hosts in which it was initially unstable. After 1000 generations in serial batch cultures under antibiotic selection for plasmid maintenance (kanamycin resistance), the stability of the mini-plasmid dramatically improved in all coevolved hosts. However, only plasmids evolved in Shewanella oneidensis showed improved stability in the ancestor, indicating that adaptive mutations had occurred in the plasmid itself. Complete genome sequence analysis of nine independently evolved plasmids showed seven unique plasmid genotypes that had various kinds of single mutations at one locus, namely, the N-terminal region of the replication initiation protein TrfA. Such parallel evolution indicates that this region was under strong selection. In five of the seven evolved plasmids, these trfA mutations resulted in a significantly higher plasmid copy number. Evolved plasmids were found to be stable in four other naive hosts, but could no longer replicate in Pseudomonas aeruginosa. This study shows that plasmids can specialize to a novel host through trade-offs between improved stability in the new host and the ability to replicate in a previously permissive host.

Several types of environmental bacteria that can aerobically degrade various aromatic compounds have been identified. The catabolic genes in these bacteria have generally been found to form operons, which promote efficient and complete degradation. However, little is known about the degradation pathways in bacteria that are difficult to culture in the laboratory. By functionally screening a metagenomic library created from activated sludge, we had earlier identified 91 fosmid clones carrying genes for extradiol dioxygenase (EDO), a key enzyme in the degradation of aromatic compounds. In this study, we analyzed 38 of these fosmids for the presence and organization of novel genes for aromatics degradation. Only two of the metagenomic clones contained complete degradation pathways similar to those found in known aromatic compound-utilizing bacteria. The rest of the clones contained only subsets of the pathway genes, with novel gene arrangements. A circular 36.7-kb DNA form was assembled from the sequences of clones carrying genes belonging to a novel EDO subfamily. This plasmid-like DNA form, designated pSKYE1, possessed genes for DNA replication and stable maintenance as well as a small set of genes for phenol degradation; the encoded enzymes, phenol hydroxylase and EDO, are capable of the detoxification of aromatic compounds. This gene set was found in 20 of the 38 analyzed clones, suggesting that this 'detoxification apparatus' may be widespread in the environment.

Plasmids are ubiquitous mobile elements that serve as a pool of many host beneficial traits such as antibiotic resistance in bacterial communities. To understand the importance of plasmids in horizontal gene transfer, we need to gain insight into the 'evolutionary history' of these plasmids, i.e. the range of hosts in which they have evolved. Since extensive data support the proposal that foreign DNA acquires the host's nucleotide composition during long-term residence, comparison of nucleotide composition of plasmids and chromosomes could shed light on a plasmid's evolutionary history. The average absolute dinucleotide relative abundance difference, termed delta-distance, has been commonly used to measure differences in dinucleotide composition, or 'genomic signature', between bacterial chromosomes and plasmids. Here, we introduce the Mahalanobis distance, which takes into account the variance-covariance structure of the chromosome signatures. We demonstrate that the Mahalanobis distance is better than the delta-distance at measuring genomic signature differences between plasmids and chromosomes of potential hosts. We illustrate the usefulness of this metric for proposing candidate long-term hosts for plasmids, focusing on the virulence plasmids pXO1 from Bacillus anthracis, and pO157 from Escherichia coli O157:H7, as well as the broad host range multi-drug resistance plasmid pB10 from an unknown host.

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.

Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.

The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: between oriV and trfA and between the tra and trb operons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1beta plasmid pBP136 and found that most Tn21Km insertions (67%) were located around oriV. A similar experiment using the oriV region of IncP-1beta plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in the oriV-trfA and tra-trb regions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between the tra and trb operons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.

Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.

The complete 41,268 bp nucleotide sequence of the IncP-1beta plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1beta backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1beta 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1beta plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.

The two haloacetate dehalogenase genes, dehH1 and dehH2, on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (TnHad1) that was flanked by two directly repeated copies of IS1071, IS1071L and IS1071R. The dehH1 gene was also flanked by IS1071L and a truncated version of IS1071 (IS1071N). TnHad1, dehH1, and IS1071N were located on a 15.6-kb class II transposon (TnHad2) whose terminal inverted repeats and res site showed high homology with those of the Tn21-related transposons. TnHad2 was defective in transposition because of its lacking the transposase and resolvase genes. TnHad2 could transpose when the Tn21-encoded transposase and resolvase were supplied in trans. These results demonstrated that Tn Had2 is a defective Tn21-related transposon carrying another class I catabolic transposon.