Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Effects of heavy metals on health risk and characteristic in surrounding atmosphere of tire manufacturing plant,Taiwan

The health and environmental effects of metal-containing carbon black (CB) particles emitted from a CB feeding area near a tire manufacturing plant were investigated. The mass ratios of PM₁ and PM_0.1 (UFPs) relative to TSP were 13.84% ± 4.88% and 50.84% ± 4.29%, respectively. The most abundant elements in all fractions were Fe, Al, and Zn. The mean percentage contributions of Al, Fe, Zn, Cu, and Co to the coarse particles ranged from 49.1% to 69.1%, thus indicating that the Al, Fe, and Zn contents in the CB particles were affected by workplace emissions. The ratios of the total mean deposition fluxes of atmospheric particle-bound heavy metals in the human respiratory tracts of workers/adults, workers/children, and adults/children were approximately 5.5, 11.0, and 2.0, respectively. The integrated risks of five elements via two exposure pathways to adults and children were 1.1 × 10⁻⁴ and 1.7 × 10⁻⁵, respectively; these numbers reflect the high cumulative carcinogenic risk posed by these toxic metals to local residents (both adults and children; limit, 10⁻⁶). These results demonstrate the potential health risk presented by particle-bound heavy metals to humans residing near tire manufacturing plants via inhalation and dermal contact exposure.

The health and environmental effects of metal-containing carbon black (CB) particles emitted from a CB feeding area near a tire manufacturing plant were investigated.     

Predicting Hospitalization for Heat-Related Illness at the Census-Tract Level: Accuracy of a Generic Heat Vulnerability Index in Phoenix,Arizona (USA)

Background

Vulnerability mapping based on vulnerability indices is a pragmatic approach for highlighting the areas in a city where people are at the greatest risk of harm from heat, but the manner in which vulnerability is conceptualized influences the results.

Objectives

We tested a generic national heat-vulnerability index, based on a 10-variable indicator framework, using data on heat-related hospitalizations in Phoenix, Arizona. We also identified potential local risk factors not included in the generic indicators.

Methods

To evaluate the accuracy of the generic index in a city-specific context, we used factor scores, derived from a factor analysis using census tract–level characteristics, as independent variables, and heat hospitalizations (with census tracts categorized as zero-, moderate-, or high-incidence) as dependent variables in a multinomial logistic regression model. We also compared the geographical differences between a vulnerability map derived from the generic index and one derived from actual heat-related hospitalizations at the census-tract scale.

Results

We found that the national-indicator framework correctly classified just over half (54%) of census tracts in Phoenix. Compared with all census tracts, high-vulnerability tracts that were misclassified by the index as zero-vulnerability tracts had higher average income and higher proportions of residents with a duration of residency < 5 years.

Conclusion

The generic indicators of vulnerability are useful, but they are sensitive to scale, measurement, and context. Decision makers need to consider the characteristics of their cities to determine how closely vulnerability maps based on generic indicators reflect actual risk of harm.

Citation

Chuang WC, Gober P. 2015. Predicting hospitalization for heat-related illness at the census-tract level: accuracy of a generic heat vulnerability index in Phoenix, Arizona (USA). Environ Health Perspect 123:606–612; http://dx.doi.org/10.1289/ehp.1307868     

An In‐House Prevocational Training Program for Newly Discharged Psychiatric Inpatients: Exploring Its Employment Outcomes and the Predictive Factors

Individuals with severe mental disorders continue to experience low employment rates. Occupational therapists play an important role in helping these individuals develop the skills and obtain the supports necessary for productive living. This retrospective cohort study aimed to explore employment outcomes and identify factors predictive of the outcomes of an in‐house prevocational training program designed for newly discharged psychiatric inpatients. Data retrieved from the files of 58 participants including demographics, diagnostic history, physical fitness, functional assessment results, the use of vocational counselling service and employment status were analyzed. The overall employment rates among the participants were high (67.2–79.3%) within the 6 months following the prevocational training program. No significant differences were found in the employment rates across the 1, 3 and 6‐month time periods post‐training. Vocational counselling service post‐training and hand function were two factors predictive of participants' employment outcomes. Occupational therapists should attend to the clients' need for continuous vocational support and carefully identify the personal, functional and environmental factors contributing to successful employment. Caution in interpreting the results is warranted because of the lack of control and randomization in this retrospective study. Additional longitudinal cohort or experimental studies would add further certainty to the current findings. Copyright © 2015 John Wiley & Sons, Ltd.     

Feasibility and diagnostic performance of hybrid PET/MRI compared with PET/CT for gynecological malignancies: a prospective pilot study

Purpose

The purpose of the study was to assess the feasibility and diagnostic performance of FDG-PET/MR imaging compared to PET/CT for staging of patients with a gynecological malignancy.

Methods

25 patients with a gynecological malignancy were prospectively enrolled into this pilot study. Patients underwent sequential full-body PET/CT and PET/MR of the abdomen and pelvis after administration of a single dose of F-18 FDG. PET/MRI and PET/CT images were independently reviewed by two expert radiologists. Readers were blinded to the results of the other imaging procedures. Clinical and pathologic information was abstracted from medical charts.

Results

18 patients were included in the final analysis with a median age of 62 years (range 31–88). 61% of patients (11/18) had cervical cancer, while the remaining patients had endometrial cancer. PET/MRI as compared to PET/CT detected all primary tumors, 7/7 patients with regional lymph nodes, and 1/1 patient with an abdominal metastasis. Two patients had additional lymph nodes outside of the abdominopelvic cavity detected on PET/CT that were not seen on PET/MRI, whereas 6 patients had parametrial invasion and one patient had invasion of the bladder seen on PET/MRI not detected on PET/CT. Five cervical cancer patients had discordant clinical vs. radiographic staging based on PET/MRI detection of soft tissue involvement. Management changed for two patients who had clinical stage IB1 and radiographic stage IIB cervical cancer.

Conclusions

PET/MRI is feasible and has at least comparable diagnostic ability to PET/CT for identification of primary cervical and endometrial tumors and regional metastases. PET/MRI may be superior to PET/CT for initial radiographic assessment of cervical cancers.

     

Lower risk of primary Sjogren’s syndrome in patients with dengue virus infection: a nationwide cohort study in Taiwan

Clinical Rheumatology - The data concerning the association between dengue viruses (DV) infection and autoimmune diseases (ADs) remain unclear and are scarce. This nationwide population-based...     

The high prevalence of Legionella pneumophila contamination in hospital potable water systems in Taiwan: implications for hospital infection control in Asia.

BACKGROUND: The major sources of Legionnaires' disease (LD) are the potable water systems of large buildings including hospitals, nursing homes, and hotels. Culturing the hospital water system for Legionella allows a preventive approach for hospital-acquired LD. However, hospital-acquired LD is rarely reported in Taiwan, and environmental cultures of Legionella in hospital water systems in Taiwan have never been systematically performed. OBJECTIVE: The objective of this study was to determine if Legionella is present in hospital water systems in Taiwan. Water quality analysis was also performed to determine if geographic differences in water quality result in different Legionella positivity rates. METHOD: The water systems of 16 hospitals throughout Taiwan were tested for Legionella by culture. Standardized culture procedures were followed. RESULTS: Legionella pneumophila was isolated from 63% (10/16) of the hospital water systems; 19% (3/16) of the hospitals had an L. pneumophila positive rate greater than 30%. L. pneumophila serogroups 1 and 6 (strains that are most responsible for Legionella infections) were isolated from 80% (8/10) and 60% (6/10), respectively, of the hospitals that yielded L. pneumophila in their water distribution systems. CONCLUSION: As was shown in epidemiological studies in the USA and Spain, hospital-acquired legionellosis may be prevalent but underdiagnosed in Taiwan.     

An adult case of Chryseobacterium meningosepticum meningitis

Chryseobacterium meningosepticum is an uncommon pathogen causing adult bacterial meningitis. Herein, we report the case history of one 21-year-old woman with this uncommon central nervous system infection. A diagnosis of adult C. meningosepticum meningitis can only be confirmed by a positive cerebrospinal fluid (CSF) culture. The patient had insulin-dependent diabetes mellitus as the underlying condition associated with this infection. The clinical presentations were fever, headache, consciousness disturbance, and seizure. CSF analysis revealed a purulent inflammatory reaction. After a 21-day course of intravenous cefepime (6 g/day) treatment, this patient was discharged in a state of complete recovery.