Temperature Sensitivity of Bacteriolysis Induced by β-Lactam Antibiotics in Amino Acid-Deprived Escherichia coli

The temperature-sensitive penicillin tolerance response previously reported in amino acid-deprived Escherichia coli (W. Kusser and E. E. Ishiguro, J. Bacteriol. 169:2310–2312, 1987) was not due to the induction of the heat shock response resulting from a temperature upshift and was therefore unrelated to the findings of another report (J. K. Powell and K. D. Young, J. Bacteriol. 173:4021–4026, 1991) indicating a positive correlation between the expression of heat shock proteins and penicillin tolerance. The thermosensitive event occurred in the lysis induction stage.     

Intergeneric Transfer of Conjugative and Mobilizable Plasmids Harbored by Escherichia coli in the Gut of the Soil Microarthropod Folsomia candida (Collembola)

The gut of the soil microarthropod Folsomia candida provides a habitat for a high density of bacterial cells (T. Thimm, A. Hoffmann, H. Borkott, J. C. Munch, and C. C. Tebbe, Appl. Environ. Microbiol. 64:2660–2669, 1998). We investigated whether these gut bacteria act as recipients for plasmids from Escherichia coli. Filter mating with E. coli donor cells and collected feces of F. candida revealed that the broad-host-range conjugative plasmid pRP4-luc (pRP4 with a luciferase marker gene) transferred to fecal bacteria at estimated frequencies of 5.4 × 10⁻¹ transconjugants per donor. The mobilizable plasmid pSUP104-luc was transferred from the IncQ mobilizing strain E. coli S17-1 and less efficiently from the IncF1 mobilizing strain NM522 but not from the nonmobilizing strain HB101. When S17-1 donor strains were fed to F. candida, transconjugants of pRP4-luc and pSUP104-luc were isolated from feces. Additionally, the narrow-host-range plasmid pSUP202-luc was transferred to indigenous bacteria, which, however, could not maintain this plasmid. Inhibition experiments with nalidixic acid indicated that pRP4-luc plasmid transfer took place in the gut rather than in the feces. A remarkable diversity of transconjugants was isolated in this study: from a total of 264 transconjugants, 15 strains belonging to the alpha, beta, or gamma subclass of the class Proteobacteria were identified by DNA sequencing of the PCR-amplified 16S rRNA genes and substrate utilization assays (Biolog). Except for Alcaligenes faecalis, which was identified by the Biolog assay, none of the isolates was identical to reference strains from data banks. This study indicates the importance of the microarthropod gut for enhanced conjugative gene transfer in soil microbial communities.Gene transfer is a process by which bacterial populations substantially increase their rates of evolution and adaptation (12, 59). Particularly, plasmid-located genes, which are transferred by conjugation from donor to recipient cells, can disseminate rapidly between even phylogenetically different bacterial groups (17, 36, 41) and microbial communities in different spatial habitats (34, 71). Such microbial genetic networks should be considered in risk assessments of releases of genetically engineered microorganisms into the environment (22, 37, 43). The probability and rate of plasmid transfer from a donor to indigenous microorganisms in a given habitat are influenced by plasmid-borne genes which determine the type of transfer mechanism (self-transmissible or mobilizable) and the host range of autonomous plasmid replication. Additionally, specific physicochemical conditions, such as temperature, water potential, and the availability of energy (substrates) for donor and recipient cells, are important factors influencing gene transfer rates in terrestrial and aquatic environments (23, 53, 64).The spread of plasmid-borne genes is still extremely difficult to predict for terrestrial habitats, since a large variety of microhabitat conditions which are not well characterized exists. In bulk soil under laboratory conditions, conjugative gene transfer from recombinant bacterial donor strains to indigenous soil bacteria has been found only under specific selective conditions or on rare occasions (11, 20, 24, 27, 50, 61). Several studies failed to detect such transfer events, and it was concluded that heterogeneity and low densities of recipient cells, as well as a lack of substrates for microbial metabolism, prevented efficient plasmid transfer in bulk soil (19, 49, 54, 75). Plant exudates increased rates of gene transfer in soil (33, 48), and higher rates of gene transfer were found in rhizospheres than in bulk soil (50, 61). It was assumed that other microsites which favor gene transfer in terrestrial habitats are associated with soil invertebrates (74). However, to date little experimental evidence to prove this assumption is available.Intraspecies transconjugants of added Enterobacter cloacae donor and recipient cells could be isolated from microcosm experiments with the variegated cutworm, Peridroma saucia, and plant material (2). The investigators in that study concluded that gene transfer events happened, most likely, in the digestive tracts or in the feces of the insects. Another recent report demonstrated that a conjugative plasmid was transferred between fed Escherichia coli strains in the guts of Rhabditis nematodes (1). Earthworms mediated transport and enhanced plasmid transfer from added donor cells to added recipients and to indigenous bacteria in soil (14, 15). High rates of intraspecies plasmid transfer, comparable to those obtained in pure broth cultures, were detected with Bacillus thuringiensis in infected lepidopterous larvae (31).Microarthropods (collembolans and mites) are the most abundant invertebrate group in the majority of soils (5) but have not been recognized, so far, for their impact on microbial gene transfer. There are some indications that microarthropods harbor a large variety of microorganisms in their guts and thereby contribute to microbial biodiversity in terrestrial environments (7, 9, 57). In the accompanying paper, we have described the gut of Folsomia candida (Collembola) as a habitat and species-specific vector for microorganisms (67). The gut of this soil-dwelling insect, which has a volume of only several nanoliters, was found to be densely colonized, predominantly by rod-shaped bacterial cells. We were interested to know whether such bacterial cells act as recipients for plasmids and thereby promote gene transfer in microbial communities. F. candida feeds, under natural conditions, on bacteria (3), fungal mycelia (6, 66), and nematodes (35). Here, we report on the results of experiments in which plasmid-bearing E. coli strains were fed to F. candida in microcosms. Self-transferable plasmids, as well as mobilizable plasmids with different host ranges, and a nonmobilizable plasmid were included in this study in order to determine the specific capacities of these different classes of plasmids to spread into indigenous bacterial populations. For detection purposes, all plasmids were engineered by the insertion of the luciferase-encoding marker gene luc or lux (30, 47).     

The role of hypersalinity in the persistence of the Texas 'brown tide' in the Laguna Madre

A brown tide bloom of the alga Aureoumbra lagunensis was presentwithout interruption in the Laguna Madre of Texas from January1990 through October 1997. This is the longest continual phytoplanktonbloom of which we are aware. Although the factors leading tothe initiation of this bloom have been well documented, thefactors contributing to its persistence are still being investigated.Two physical characteristics of the Laguna Madre may play animportant role: the long turnover time for waters in this coastallagoon (–1 year) and the hypersaline conditions that usuallyexist (40–60 PSU) due to evaporation exceeding precipitation.In this study, we examined the effects of salinity on the growthrates of the brown tide alga and on the growth of one of itsprotozoan grazers. Historical data from before the onset ofthe brown tide provide evidence for the suppression of microzooplanktonpopulations and mesozooplankton growth caused by hypersalinity.The brown tide alga will grow in a remarkably wide range ofsalinities ranging from 10 to 90 PSU. Maximum growth rates areachieved at salinities ranging from 20 to 60 PSU. One commongrazer on the brown tide alga, the heterotrophic dinoftagellateOxyrrhis marina, was found to grow more slowly under hypersalineconditions. The normally hypersaline conditions of the LagunaMadre may, therefore, favor the brown tide alga over other phytoplanktonspecies that do not grow well under hypersaline conditions,and also suppress the growth and feeding rates of potentialgrazers.     

Defining the physician's duty to warn: consensus statement of Ontario's Medical Expert Panel on Duty to Inform

Ontario''s Medical Expert Panel on Duty to Inform was formed to consider the duty of Ontario physicians in circumstances where a patient threatens to kill or cause serious bodily harm to a third party. The panel was concerned about the implications of any duty to inform on the integrity of the physician-patient relationship, particularly with respect to confidentiality. The panel agreed that regulations safeguarding the confidentiality of patient information ought to be changed only if there is a critical reason for doing so, but, after deliberation, the panel members concluded that the need to protect the public from serious risk of harm is a paramount concern that should supersede the duty of confidentiality. The recommendations reported here were endorsed in principle by the panelists and the groups they represented (the Royal College of Physicians and Surgeons of Canada, the Canadian Medical Protective Association, the College of Physicians and Surgeons of Ontario, the Ontario College of Family Physicians and the Ontario Medical Association) and are being implemented by the College of Physicians and Surgeons of Ontario.     

Alkoxide-catalyzed ring-opening of a novel homosaccharin derivative: synthesis of potent, selective P3-lactam thrombin inhibitors containing P4-o-alkoxycarbonylbenzylsulfonamide residues

A series of lactam derivatives 1b-g featuring P₄-o-alkoxycarbonylbenzylsulfonamide residues along with the potential P₄-homosaccharin prodrug candidate 1h was prepared in order to probe the thrombin S₃ specificity pocket. The synthesis and alkoxide-catalyzed ring opening of the novel homosaccharin intermediate 7 followed by subsequent elaboration delivered the targets 1b-h which were potent and selective thrombin inhibitors. The design, synthesis, and biological activity of these targets will be presented.     

Enhanced neointimal growth in cultured rabbit aorta following in vivo balloon angioplasty

Summary We have used in vivo balloon catheterization in combination with in vitro organ culture to develop a model system for vascular neointima formation. A Fogarty balloon catheter was used to deendothelialize and rupture the internal elastic lamina of aortae in adult rabbits. After three d of recovery, aortae were harvested, divided into segments, and placed into organ culture. We obtained a daily index of cell proliferation in cultured vessels using [³H]thymidine incorporation into DNA. Also, segments were collected and processed for routine histology or immunohistochemistry. Aortic segments that had undergone ballooning 3 d before harvest and then cultured exhibited diffuse neointimal growth after several d in vitro, whereas those from sham-operated (nonballooned) rabbits showed generally only a single endothelial cell layer that is characteristic of normal intima. Aortae that were harvested, balloon-damaged in vitro, and then cultured exhibited no neointimal growth. The neointima that developed in cultured segments from in vivo ballooned rabbits was primarily of smooth muscle cell origin as determined by positive immunostaining for α-smooth muscle actin. The intima:media thickness ratios were significantly higher in aortic segments from ballooned rabbits at harvest and after 4 or 7 d in culture compared with those from nonballooned rabbits. Also, the [³H]thymidine index was higher in the in vivo ballooned aorta compared to non-ballooned or in vitro ballooned vessel. We conclude that ballooning in vivo followed by exposure to blood-borne elements produces an enhanced proliferative response in cultured vessels that is distinct from other in vitro models of neointimal growth.     

Technical and Policy Challenges in Developing and Implementing Risk-Based Environmental Regulations

The reform in environmental regulations being considered at both federal and state levels is intended to enhance the value and effectiveness of a rule by incorporating risk assessment and cost benefit analysis in the rule making process or regulatory implementation. Although a risk based approach may not provide a panacea to all environmental problem solving, it offers some obvious advantages over the status quo. In particular, it establishes a scientifically defensible basis for evaluating the trade off between risks, costs and benefits in making prudent environmental decisions and developing effective regulatory policies. This paper presents a conceptual framework for risk reduction, summarizes the current status in risk-based legislation at the federal level, provides examples of how various states are using risk based approaches in their regulatory programs, addresses aspects of technical and policy challenges in rule making and other policy and enforcement decisions and provides suggestions for meeting these challenges.