Diversity and Dynamics of Indigenous \u3cem\u3eRhizobium japonicum\u3c/em\u3e Populations

A simple method, based upon the separation of cellular proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has been devised for distinguishing between isolates of Rhizobium japonicum. Eleven laboratory strains, previously classified into five serogroups, were analyzed by gel electrophoresis. Groups determined subjectively according to protein patterns matched the serogroups, with one exception. Most strains within serogroups could be distinguished from one another. For studying the ecology of Rhizobium, an important advantage of this technique compared with serology or phage typing is that it discriminates among previously unencountered indigenous bacterial isolates as well as among known laboratory strains. SDS-gels were used to analyze the Rhizobium population of 500 nodules, sampled throughout the growing season, from soybeans at two different Wisconsin localities. Although the soybeans had been inoculated with laboratory strains of R. japonicum, indigenous R. japonicum predominated. At one location, 19 indigenous gel types were distinguished and classified mainly into four groups. At the other location, 18 gel types, falling mainly into three groups, were detected. The predominance of a particular group varied, in some cases dramatically, depending upon the time and depth of nodule formation

Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB

Genetic Basis for \u3cem\u3eRhizobium etli\u3c/em\u3e CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis

Infection of Soybean and Pea Nodules by \u3cem\u3eRhizobium\u3c/em\u3e spp. Purine Auxotrophs in the Presence of 5-aminoimidazole-4-Carboxamide Riboside

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth

\u3cem\u3eRhizobium japonicum\u3c/em\u3e Mutants Defective in Symbiotic Nitrogen Fixation

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads

Stretching, embeddedness, and scripts in a sociotechnical transition: explaining the failure of electric mobility at Better Place (2007–2013)

Based on field research, interviews, and participant observation, this study explores the failure of Better Place—a now bankrupt company—to successfully demonstrate and deploy battery swapping stations and electric vehicle charging infrastructure. To do so, it draws from concepts in innovation studies, sociotechnical transitions, management science, organizational studies, and sociology. The study expands upon the notion of “fit-stretch”, which explains how innovations can move from an initial “fit” (with existing user practices, discourses, technical form) to a subsequent “stretch” (as the technology further develops, new functionalities are opened up, etc.) in the process of long-term transitions. It also draws from the “dialectical issue life cycle model” or “triple embeddedness framework” to explain the process whereby incumbent industry actors can introduce defensive innovations to “contain” a new niche from expanding. It lastly incorporates elements from design-driven innovation and organizational learning related to schemas and scripts, concepts that illustrate the vision-dependent and discursive elements of the innovation process. It uses the case study of Better Place to test and build upon these concepts. With a market valuation of more than $2 billion, Better Place was poised to become one of the most innovative companies in the electric mobility market. Yet after operating for five years it declared bankruptcy and saw its assets sold off for less than$500,000. We suggest here that Better Place failed because it “stretched” to the point that it “broke;” that it provoked a defensive response from both old automotive manufacturers (such as General Motors) and new ones (such as Tesla); and that the fantastic nature of its visionary scripts convinced its investors and promoters to unrealistically raise expectations and downplay persistent risks

Does the Sun Shrink with Increasing Magnetic Activity?

We have analyzed the full set of SOHO/MDI f- and p-mode oscillation frequencies from 1996 to date in a search for evidence of solar radius evolution during the rising phase of the current activity cycle. Like Antia et al. (2000), we find that a significant fraction of the f-mode frequency changes scale with frequency; and that if these are interpreted in terms of a radius change, it implies a shrinking sun. Our inferred rate of shrinkage is about 1.5 km/y, which is somewhat smaller than found by Antia et al. We argue that this rate does not refer to the surface, but rather to a layer extending roughly from 4 to 8 Mm beneath the visible surface. The rate of shrinking may be accounted for by an increasing radial component of the rms random magnetic field at a rate that depends on its radial distribution. If it were uniform, the required field would be ~7 kG. However, if it were inwardly increasing, then a 1 kG field at 8 Mm would suffice. To assess contribution to the solar radius change arising above 4Mm, we analyzed the p-mode data. The evolution of the p-mode frequencies may be explained by a magnetic^M field growing with activity. The implications of the near-surface magnetic field changes depend on the anisotropy of the random magnetic field. If the field change is predominantly radial, then we infer an additional shrinking at a rate between 1.1-1.3 km/y at the photosphere. If on the other hand the increase is isotropic, we find a competing expansion at a rate of 2.3 km/y. In any case, variations in the sun's radius in the activity cycle are at the level of 10^{-5} or less, hence have a negligible contribution to the irradiance variations.Comment: 10 pages (ApJ preprint style), 4 figures; accepted for publication in Ap

Facilitators and barriers to the successful implementation of pediatric antibacterial drug trials: Findings from CTTI's survey of investigators.

An urgent need exists to develop new antibacterial drugs for children. We conducted research with investigators of pediatric antibacterial drug trials to identify facilitators and barriers in the conduct of these trials. Seventy-three investigators completed an online survey assessing the importance of 15 facilitators (grouped in 5 topical categories) and the severity of 36 barriers (grouped in 6 topical categories) to implementing pediatric antibacterial drug trials. Analysis focused on the identification of key factors that facilitate the successful implementation of pediatric antibacterial drug trials and the key barriers to implementation. Almost all investigators identified two factors as very important facilitators: having site personnel for enrollment and having adequate funding. Other top factors were related to staffing. Among the barriers, factors related to parent concerns and consent were prominent, particularly obtaining parental consent when there was disagreement between parents, concerns about the number of blood draws, and concerns about the number of invasive procedures. Having overly narrow eligibility criteria was also identified as a major barrier. The survey findings suggest three areas in which to focus efforts to help facilitate ongoing drug development: (1) improving engagement with parents of children who may be eligible to enroll in a pediatric antibacterial drug trial, (2) broadening inclusion criteria to allow more participants to enroll, and (3) ensuring adequate staffing and establishing sustainable financial strategies, such as funding pediatric trial networks. The pediatric antibacterial drug trials enterprise is likely to benefit from focused efforts by all stakeholders to remove barriers and enhance facilitation